Polynucleotide encoding autoantigens associated with endometriosis

ABSTRACT

This invention provides a polynucleotide encoding Repro-EN-1.0 and IB1, polypeptides associated with endometriosis. Auto-antibodies against Repro-EN-1.0 and IB1 have been found in subjects diagnosed with endometriosis. This invention also provides methods of using this polynucleotide and polypeptide.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to co-pending application ofSchneider et al., “Diagnosis Of Autoimmune Disease By Detecting IgE orIgG₄ Autoantibodies Against Autoantigens,” attorney docket no.018002-001200, filed on even date herewith, the content of which isincorporated herein by reference in its entirety.

[0002] This application is a continuation in part of U.S. patentapplication Ser. No. 09/359,084 filed Jul. 22, 1999, which, in turn, isa continuation of Provisional Patent Application Serial No. 60/094,930,filed Jul. 31, 1998.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] Certain work described herein was supported by SBIR grant no.1R43 HD 33022-01A2 between the United States Department of Health andHuman Services and Reprogen, Inc. The Government may have certain rightsin this invention.

FIELD OF THE INVENTION

[0004] This invention is directed to the field of molecular biology ingeneral, and, more specifically, to a polypeptide associated withendometriosis, an isolated polynucleotide encoding the polypeptide, andmethods of using these molecules.

BACKGROUND OF THE INVENTION

[0005] Endometriosis is a painful disorder that is characterized by theectopic implantation of functioning endometrial tissue into theabdominal wall and the outer surface of various organs including, mostcommonly, the lower bowel, ovaries and fallopian tubes. P. Vigano et al.(1991) Fertility and Sterility 56:894. Currently, endometriosis-specificgenes have not been identified and the events relating to thedevelopment of endometriosis are poorly understood. However, severalreports suggest that retrograde menstruation linked with abnormal immunefunction may play a role in establishing ectopic endometrium lesions. T.Ishimaru and H. Masuzaki (1991) Am. J. Obstet. Gynecol. 165:210-214.Many attempts to isolate antigens from ectopic endometrium lesions havefailed, due to the necrotic nature of the lesions.

[0006] Endometriosis also is recognized has having an autoimmunecomponent. IgG and IgA auto-antibodies that react with multipleendometrial antigens have been documented in patients withendometriosis. However, attempts to develop IgG or IgA-based assays forthe diagnosis of endometriosis has fallen short of fruition. S.Fernandez-Shaw et al., (1996) Hum. Reprod. 11:180-1184. R. A. Wild etal. (1991) Obstetrics and Gynecology 77:927. Studies have shown thatcirculating IgG antibodies that bind multiple endometrial proteins canbe detected in women with endometriosis to varying degrees. Thirty-fivepercent to 74% of patients have sera reactive with endometrial proteins.O. Odukoya et al. (1996) Acta Obstet. Gynecol. Scand. 75:927-931; J. G.Kim et al. (1995) Am. J. Reprod. Immunol. 34:80-87; O. A. Odukoya et al.(1995) Hum. Reprod. 10: 1214-1219. It has also been shown thatendometrial antibody titers in patients that respond well to danazol aresignificantly lower ({fraction (7/18)} (39%) treated patients hadelevated titers) than those patients with untreated endometriosis orpatients that responded poorly to treatment ({fraction (17/23)} (74%)untreated patients had elevated titers). A. El-Roeiy et al. (1988)Fertility and Sterility 50:864-871; H. J. Chihal et al. (1986) Fertilityand Sterility 46:408-411. In addition, it has been recently reportedthat women with endometriosis have elevated levels of IL-4, a Th2mediating cytokine, and that treatment with danazol reduces the levelsof IL-4 in women that respond well to treatment. C. -C. Hsu et al.(1997) Fertility and Sterility 67:1059-1064.

SUMMARY OF THE INVENTION

[0007] This invention provides an isolated cDNA molecule and analternately spliced variant encoding autoantigens associated withendometriosis. The autoantigen is called Repro-EN-1.0. The alternatelyspliced variant is called IB1. Subjects diagnosed with endometriosishave been found to have antibodies that specifically bind toRepro-EN-1.0 polypeptide and/or a IB1 polypeptide. These antibodiesrepresent a highly sensitive and specific diagnostic marker forendometriosis. Recombinant Repro-EN-1.0 protein and recombinant IB1protein are useful to detect such antibodies in immunoassays.

[0008] In one aspect this invention provides a recombinantpolynucleotide comprising a nucleotide sequence encoding a polypeptideepitope of at least 5 amino acids of Repro-EN-1.0 (SEQ ID NO:2), or IB1(SEQ ID NO:4) wherein the epitope specifically binds to antibodies fromsubjects diagnosed with endometriosis. In one embodiment the nucleotidesequence is selected from the Repro-EN-1.0 sequence of SEQ ID NO:1 orIB1 sequence of SEQ ID NO: 3. In another embodiment the nucleotidesequence is identical to nucleotides 176 to 2755 of SEQ ID NO:1 ornucleotides 176 to 2986 of SEQ ID NO:3. In another embodiment thepolynucleotide further comprises an expression control sequenceoperatively linked to the nucleotide sequence.

[0009] In another aspect this invention provides a polynucleotide primerpair which amplifies a nucleotide sequence encoding a polypeptideepitope of at least 5 amino acids of Repro-EN-1.0, or IB1 wherein theepitope specifically binds to antibodies from subjects diagnosed withendometriosis. The pair comprises: 1) a 3′ primer of at least 7nucleotides that specifically hybridizes to a 3′ end of the nucleotidesequence or downstream from the sequence, and 2) a 5′ primer of at least7 nucleotides that specifically hybridizes to the 3′ end of thecomplement of the nucleotide sequence or downstream from the complementof the sequence.

[0010] In another aspect this invention provides a recombinant cellcomprising a recombinant polynucleotide comprising an expression controlsequence operatively linked to a nucleotide sequence encoding apolypeptide epitope of at least 5 amino acids of Repro-EN-1.0 (SEQ IDNO:2), or IB1 (SEQ ID NO:4), wherein the epitope specifically binds toantibodies from subjects diagnosed with endometriosis.

[0011] In another aspect this invention provides a method for detectinga target polynucleotide comprising a nucleotide sequence selected fromRepro-EN-1.0 cDNA (SEQ ID NO:1) or its complement, or IB1 cDNA (SEQ IDNO:3) or its complement in a sample. The method comprises the steps of:(a) contacting the sample with a polynucleotide probe or primercomprising a sequence of at least 7 nucleotides that specificallyhybridizes to the nucleotide sequence and (b) detecting whether theprobe or primer has specifically hybridized to the targetpolynucleotide, whereby specific hybridization provides a detection ofthe target polynucleotide in the sample.

[0012] In another aspect this invention provides a purified, recombinantRepro-EN-1.0 polypeptide whose amino acid sequence is identical to thatof SEQ ID NO:2, or an allelic variant of SEQ ID NO:2, or an IB1polypeptide whose amino acid sequence is identical to that of SEQ IDNo:4, or an allelic variant of SEQ ID No:4.

[0013] In another aspect this invention provides a purified polypeptidecomprising an epitope of at least 5 amino acids of Repro-EN-1.0 (SEQ IDNO:2), or an epitope of at least 5 amino acids of IB1 (SEQ ID NO:4),wherein the epitope specifically binds to antibodies from subjectsdiagnosed with endometriosis.

[0014] In another aspect this invention provides a compositionconsisting essentially of an antibody that specifically binds toRepro-EN-1.0 polypeptide (SEQ ID NO:2), or IB1 polypeptide (SEQ IDNO:4).

[0015] In another aspect this invention provides a method for detectinga Repro-EN-1.0 polypeptide or IB1 polypeptide in a sample. The methodcomprises the steps of: (a) contacting the sample with a ligand thatspecifically binds to the Repro-EN-1.0 polypeptide or IB1 polypeptideand (b) detecting specific binding between the ligand and Repro-EN-1.0polypeptide or IB1 polypeptide. Specific binding provides a detection ofRepro-EN-1.0 polypeptide or IB1 polypeptide in the sample. In oneembodiment, the ligand is an antibody.

[0016] In another aspect this invention provides a method diagnosingendometriosis in a subject. The method comprises the steps of: (a)detecting a test amount of an antibody that specifically binds toRepro-EN-1.0 polypeptide or IB1 polypeptide in a sample from thesubject; and (b) comparing the test amount with a normal range of theantibody in a control sample from a subject who does not suffer fromendometriosis. A test amount above the normal range provides a positiveindication in the diagnosis of endometriosis. The sample can be a bloodproduct, e.g., serum, peritoneal fluid, menstrual fluid, vaginalsecretion or urine. In one embodiment the antibody is an IgE, IgG orIgG₄ immunoglobulin. In another embodiment the step of detectingcomprises capturing the antibody from the sample with an immobilizedRepro-EN-1.0 or a peptide comprising an epitope of Repro-EN-1.0 or IB1or a peptide comprising an epitope of IB1, and detecting capturedantibody. The step of detecting captured antibody can comprisecontacting the captured antibody with a detectable antibody thatspecifically binds immunoglobulins and detecting binding between thecaptured antibody and the detectable antibody. In another embodiment thestep of detecting can comprise capturing the antibody from the samplewith an immobilized anti-immunoglobulin antibody and detecting capturedantibody. The step of detecting captured antibody can comprisecontacting the captured antibody with Repro-EN-1.0 or a polypeptide, orIB1 or a peptide comprising an epitope of IB1, comprising an epitope ofRepro-EN-1.0 and detecting binding between the captured antibody and theRepro-EN-1.0 or polypeptide or IB1 or polypeptide.

[0017] In another aspect this invention provides a method for use infollowing the progress of endometriosis in a subject. The methodcomprises the steps of: (a) detecting first and second amounts of anantibody that specifically bind Repro-EN-1.0 polypeptide, or IB1polypeptide, in samples from the subject at a first and a second time,respectively; and (b) comparing the first and second amounts. Anincrease between the first and second amounts indicates progression ofthe endometriosis and a decrease between the first and second amountsindicates remission of the endometriosis.

[0018] In another aspect this invention provides an isolated MHC-peptidecomplex comprising at least a portion of an MHC Class I molecule or anMHC Class II molecule, wherein the portion comprises a binding site thatspecifically binds a peptide having an amino acid binding motif specificto the molecule, and wherein the portion engages in CD4-mediated orCD8-mediated binding to T cells, and a peptide of at least 8 amino acidsin a sequence selected from the amino acid sequence of Repro-EN-1.0 (SEQID NO:2) or IB1 (SEQ ID NO:4), wherein the peptide comprises the aminoacid binding motif and comprises an epitope that specifically binds to aT cell receptor; wherein the complex specifically binds a T cell havinga T cell receptor that specifically binds to the epitope, and whereinspecific binding induces anergy in the T cell.

[0019] In another aspect this invention provides a method for treatingendometriosis in a subject comprising the step of inhibiting an immuneresponse against Repro-EN-1.0 or IB1 in the subject. The method cancomprise administering to the subject an immunosuppressant in an amounteffective to inhibit the immune response, administering to the subjectan isolated MHC-peptide complex of the invention in an amount effectiveto inhibit the immune response or administering to the subject ananti-idiotypic antibody that specifically binds to an antigen bindingsite of an antibody that specifically binds to Repro-EN-1.0 or IB1 in anamount effective to inhibit the immune response.

[0020] In another aspect this invention provides a screening method fordetermining whether a compound increases or decreases the expression ofRepro-EN-1.0 in a cell comprising contacting the cell with the compoundand determining whether the production of Repro-EN-1.0 mRNA orpolypeptide, or IB1 mRNA or polypeptide, are increased or decreased.

[0021] In another aspect this invention provides a method of detecting achromosomal translocation of a Repro-EN-1.0 gene or IB1 gene comprisingthe steps of: a) hybridizing a labeled polynucleotide probe thatspecifically hybridizes with the Repro-EN-1.0 nucleotide sequence of SEQID NO:1 or its complement, or IB1 nucleotide sequence of SEQ ID NO:3 orits complement, to a chromosome spread from a cell sample to determinethe pattern of hybridization and b) determining whether the pattern ofhybridization differs from a normal pattern. A difference in the patternprovides detection of a translocation.

[0022] In another aspect this invention provides a method of detectingpolymorphic forms of Repro-EN-1.0 or IB1 comprising the steps of: a)determining the identity of a nucleotide or amino acid at a selectedposition within the sequence of a test Repro-EN-1.0 gene or polypeptide,or IB1 gene or polypeptide; b) determining the identity of thenucleotide or amino acid at the corresponding position of nativeRepro-EN-1.0 (SEQ ID NO:1 or 2) gene or polypeptide, or IB1 (SEQ No:3 or4) gene or polypeptide; and c) comparing the identity from the test geneor polynucleotide with the identity of the native gene or polypeptide,whereby a difference in identity indicates that the test polynucleotideis a polymorphic form of Repro-EN-1.0 or IB1.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a northern blot analysis of Repro-EN-1.0 expression invarious tissues.

[0024]FIG. 2 is a northern blot analysis of Repro-EN-1.0 expressioncomparing various normal v. cancerous tissues.

[0025]FIG. 3 is a northern blot analysis of Repro-EN-1.0 expression invarious tissue culture cells.

DETAILED DESCRIPTION OF THE INVENTION

[0026] I. Definitions

[0027] Unless defined otherwise, all technical and scientific terms usedherein have the meaning commonly understood by a person skilled in theart to which this invention belongs. The following references provideone of skill with a general definition of many of the terms used in thisinvention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

[0028] “Polynucleotide” refers to a polymer composed of nucleotideunits. Polynucleotides include naturally occurring nucleic acids, suchas deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. Nucleic acid analogs include those which includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor which include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.” “cDNA” refers to a DNA that iscomplementary or identical to an mRNA, in either single stranded ordouble stranded form.

[0029] Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand having the same sequence as an mRNAtranscribed from that DNA and which are located 5′ to the 5′-end of theRNA transcript are referred to as “upstream sequences”; sequences on theDNA strand having the same sequence as the RNA and which are 3′ to the3′ end of the coding RNA transcript are referred to as “downstreamsequences.”

[0030] “Complementary” refers to the topological compatibility ormatching together of interacting surfaces of two polynucleotides. Thus,the two molecules can be described as complementary, and furthermore,the con-tact surface characteristics are complementary to each other. Afirst polynucleotide is complementary to a second polynucleotide if thenucleotide sequence of the first polynucleotide is identical to thenucleotide sequence of the polynucleotide binding partner of the secondpolynucleotide. Thus, the polynucleotide whose sequence 5′-TATAC-3′ iscomplementary to a polynucleotide whose sequence is 5′-GTATA-3′.

[0031] A nucleotide sequence is “substantially complementary” to areference nucleotide sequence if the sequence complementary to thesubject nucleotide sequence is substantially identical to the referencenucleotide sequence.

[0032] “Encoding” refers to the inherent property of specific sequencesof nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA,to serve as templates for synthesis of other polymers and macromoleculesin biological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

[0033] “Recombinant polynucleotide” refers to a polynucleotide havingsequences that are not naturally joined together. An amplified orassembled recombinant polynucleotide may be included in a suitablevector, and the vector can be used to transform a suitable host cell. Ahost cell that comprises the recombinant polynucleotide is referred toas a “recombinant host cell.” The gene is then expressed in therecombinant host cell to produce, e.g., a “recombinant polypeptide.” Arecombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

[0034] “Expression control sequence” refers to a nucleotide sequence ina polynucleotide that regulates the expression (transcription and/ortranslation) of a nucleotide sequence operatively linked thereto.“Operatively linked” refers to a functional relationship between twoparts in which the activity of one part (e.g., the ability to regulatetranscription) results in an action on the other part (e.g.,transcription of the sequence). Expression control sequences caninclude, for example and without limitation, sequences of promoters(e.g., inducible or constitutive), enhancers, transcription terminators,a start codon (i.e., ATG), splicing signals for introns, and stopcodons.

[0035] “Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and virusesthat incorporate the recombinant polynucleotide.

[0036] “Amplification” refers to any means by which a polynucleotidesequence is copied and thus expanded into a larger number ofpolynucleotide molecules, e.g., by reverse transcription, polymerasechain reaction, and ligase chain reaction.

[0037] “Primer” refers to a polynucleotide that is capable ofspecifically hybridizing to a designated polynucleotide template andproviding a point of initiation for synthesis of a complementarypolynucleotide. Such synthesis occurs when the polynucleotide primer isplaced under conditions in which synthesis is induced, i.e., in thepresence of nucleotides, a complementary polynucleotide template, and anagent for polymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

[0038] “Probe,” when used in reference to a polynucleotide, refers to apolynucleotide that is capable of specifically hybridizing to adesignated sequence of another polynucleotide. A probe specificallyhybridizes to a target complementary polynucleotide, but need notreflect the exact complementary sequence of the template. In such acase, specific hybridization of the probe to the target depends on thestringency of the hybridization conditions. Probes can be labeled with,e.g., chromogenic, radioactive, or fluorescent moieties and used asdetectable moieties.

[0039] A first sequence is an “antisense sequence” with respect to asecond sequence if a polynucleotide whose sequence is the first sequencespecifically hybridizes with a polynucleotide whose sequence is thesecond sequence.

[0040] “Hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refers to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

[0041] The term “stringent conditions” refers to conditions under whicha probe will hybridize preferentially to its target subsequence, and toa lesser extent to, or not at all to, other sequences. “Stringenthybridization” and “stringent hybridization wash conditions” in thecontext of nucleic acid hybridization experiments such as Southern andnorthern hybridizations are sequence dependent, and are different underdifferent environmental parameters. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,N.Y. Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the Tm for a particular probe.

[0042] An example of stringent hybridization conditions forhybridization of complementary nucleic acids which have more than 100complementary residues on a filter in a Southern or northern blot is 50%formalin with 1 mg of heparin at 42° C., with the hybridization beingcarried out overnight. An example of highly stringent wash conditions is0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent washconditions is a 0.2× SSC wash at 65° C. for 15 minutes (see, Sambrook etal. for a description of SSC buffer). Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Anexample medium stringency wash for a duplex of, e.g., more than 100nucleotides, is 1× SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6× SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

[0043] “Polypeptide” refers to a polymer composed of amino acidresidues, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides.

[0044] Conventional notation is used herein to portray polypeptidesequences: the left-hand end of a polypeptide sequence is theamino-terminus; the right-hand end of a polypeptide sequence is thecarboxyl-terminus.

[0045] “Conservative substitution” refers to the substitution in apolypeptide of an amino acid with a functionally similar amino acid. Thefollowing six groups each contain amino acids that are conservativesubstitutions for one another:

[0046] 1) Alanine (A), Serine (S), Threonine (T);

[0047] 2) Aspartic acid (D), Glutamic acid (E);

[0048] 3) Asparagine (N), Glutamine (O);

[0049] 4) Arginine (R), Lysine (K);

[0050] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0051] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0052] “Allelic variant” refers to any of two or more polymorphic formsof a gene occupying the same genetic locus. Allelic variations arisenaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequences. “Allelic variants” also refer to cDNAs derived from mRNAtranscripts of genetic allelic variants, as well as the proteins encodedby them.

[0053] Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

[0054] The terms “identical” or percent “identity,” in the context oftwo or more polynucleotide or polypeptide sequences, refer to two ormore sequences or sub-sequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

[0055] The phrase “substantially identical,” in the context of twonucleic acids or polypeptides, refers to two or more sequences orsub-sequences that have at least 60%, 80%, 90%, 95% or 98% nucleotide oramino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In a mostpreferred embodiment, the sequences are substantially identical over theentire length of the coding regions.

[0056] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

[0057] Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

[0058] One example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

[0059] Another example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word-length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word-length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Nat'l. Acad. Sci. USA 89:10915 (1989)).

[0060] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Proc. Natl. Acad.Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

[0061] A further indication that two nucleic acid sequences orpolypeptides are substantially identical is that the polypeptide encodedby the first nucleic acid is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described herein.

[0062] An “affinity agent” is a compound that specifically ornon-specifically binds to a target molecule. Affinity agents thatnon-specifically bind to a molecule include, for example, anion orcation exchange resins, or materials that bind hydrophobic orhydrophilic molecules, or metal ions.

[0063] A “ligand” is a compound that specifically binds to a targetmolecule.

[0064] A “receptor” is compound that specifically binds to a ligand.

[0065] “Antibody” refers to a polypeptide ligand substantially encodedby an immunoglobulin gene or immunoglobulin genes, or fragments thereof,which specifically binds and recognizes an epitope (e.g., an antigen).The recognized immunoglobulin genes include the kappa and lambda lightchain constant region genes, the alpha, gamma, delta, epsilon and muheavy chain constant region genes, and the myriad immunoglobulinvariable region genes. Antibodies exist, e.g., as intact immunoglobulinsor as a number of well characterized fragments produced by digestionwith various peptidases. This includes, e.g., Fab′ and F(ab)′2fragments. The term “antibody,” as used herein, also includes antibodyfragments either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA methodologies. It alsoincludes polyclonal antibodies, monoclonal antibodies, chimericantibodies and humanized antibodies. “Fc” portion of an antibody refersto that portion of an immunoglobulin heavy chain that comprises one ormore heavy chain constant region domains, CH1, CH2 and CH3, but does notinclude the heavy chain variable region.

[0066] A ligand or a receptor (e.g., an antibody) “specifically bindsto” or “is specifically immunoreactive with” a compound analyte when theligand or receptor functions in a binding reaction which isdeterminative of the presence of the analyte in a sample ofheterogeneous compounds. Thus, under designated assay (e.g.,immunoassay) conditions, the ligand or receptor binds preferentially toa particular analyte and does not bind in a significant amount to othercompounds present in the sample. For example, a polynucleotidespecifically binds under hybridization conditions to an analytepolynucleotide comprising a complementary sequence; an antibodyspecifically binds under immunoassay conditions to an antigen analytebearing an epitope against which the antibody was raised; and anadsorbent specifically binds to an analyte under proper elutionconditions.

[0067] “Immunoassay” refers to a method of detecting an analyte in asample in which specificity for the analyte is conferred by the specificbinding between an antibody and a ligand. This includes detecting anantibody analyte through specific binding between the antibody and aligand. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, New York, for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

[0068] “Vaccine” refers to an agent or composition containing an agenteffective to confer a therapeutic degree of immunity on an organismwhile causing only very low levels of morbidity or mortality. Methods ofmaking vaccines are, of course, useful in the study of the immune systemand in preventing and treating animal or human disease.

[0069] An “immunogenic amount” is an amount effective to elicit animmune response in a subject.

[0070] “Substantially pure” or “isolated” means an object species is thepredominant species present (i.e., on a molar basis, more abundant thanany other individual macromolecular species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50% (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition means that about 80% to 90% or more of the macromolecularspecies present in the composition is the purified species of interest.The object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) if the composition consists essentially of a singlemacromolecular species. Solvent species, small molecules (<500 Daltons),stabilizers (e.g., BSA), and elemental ion species are not consideredmacromolecular species for purposes of this definition.

[0071] “Naturally-occurring” as applied to an object refers to the factthat the object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring.

[0072] “Detecting” refers to determining the presence, absence, oramount of an analyte in a sample, and can include quantifying the amountof the analyte in a sample or per cell in a sample.

[0073] “Detectable moiety” or a “label” refers to a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means. For example, useful labels include ³²P, ³⁵S,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteinsfor which antisera or monoclonal antibodies are available, or nucleicacid molecules with a sequence complementary to a target. The detectablemoiety often generates a measurable signal, such as a radioactive,chromogenic, or fluorescent signal, that can be used to quantitate theamount of bound detectable moiety in a sample. The detectable moiety canbe incorporated in or attached to a primer or probe either covalently,or through ionic, van der Waals or hydrogen bonds, e.g., incorporationof radioactive nucleotides, or biotinylated nucleotides that arerecognized by streptavadin. The detectable moiety may be directly orindirectly detectable. Indirect detection can involve the binding of asecond directly or indirectly detectable moiety to the detectablemoiety. For example, the detectable moiety can be the ligand of abinding partner, such as biotin, which is a binding partner forstreptavadin, or a nucleotide sequence, which is the binding partner fora complementary sequence, to which it can specifically hybridize. Thebinding partner may itself be directly detectable, for example, anantibody may be itself labeled with a fluorescent molecule. The bindingpartner also may be indirectly detectable, for example, a nucleic acidhaving a complementary nucleotide sequence can be a part of a branchedDNA molecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules. (See, e.g., P D. Fahrlander and A.Klausner, Bio/Technology (1988) 6:1165.) Quantitation of the signal isachieved by, e.g., scintillation counting, densitometry, or flowcytometry.

[0074] “Linker” refers to a molecule that joins two other molecules,either covalently, or through ionic, van der Waals or hydrogen bonds,e.g., a nucleic acid molecule that hybridizes to one complementarysequence at the 5′ end and to another complementary sequence at the 3′end, thus joining two non-complementary sequences.

[0075] “Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in a mammal. A pharmaceutical composition comprises apharmacologically effective amount of an active agent and apharmaceutically acceptable carrier. “Pharmacologically effectiveamount” refers to that amount of an agent effective to produce theintended pharmacological result. “Pharmaceutically acceptable carrier”refers to any of the standard pharmaceutical carriers, buffers, andexcipients, such as a phosphate buffered saline solution, 5% aqueoussolution of dextrose, and emulsions, such as an oil/water or water/oilemulsion, and various types of wetting agents and/or adjuvants. Suitablepharmaceutical carriers and formulations are described in Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995).Preferred pharmaceutical carriers depend upon the intended mode ofadministration of the active agent. Typical modes of administrationinclude enteral (e.g., oral) or parenteral (e.g., subcutaneous,intramuscular, intravenous or intraperitoneal injection; or topical,transdermal, or transmucosal administration). A “pharmaceuticallyacceptable salt” is a salt that can be formulated into a compound forpharmaceutical use including, e.g., metal salts (sodium, potassium,magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0076] “Small organic molecule” refers to organic molecules of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes organic biopolymers (e.g., proteins, nucleic acids,etc.). Preferred small organic molecules range in size up to about 5000Da, up to about 2000 Da, or up to about 1000 Da.

[0077] A “subject” of diagnosis or treatment is a human or non-humanmammal. Non-human mammals subject to diagnosis or treatment include, forexample, primates, ungulates, canines and felines.

[0078] “Treatment” refers to prophylactic treatment or therapeutictreatment.

[0079] A “prophylactic” treatment is a treatment administered to asubject who does not exhibit signs of a disease or exhibits only earlysigns for the purpose of decreasing the risk of developing pathology.

[0080] A “therapeutic” treatment is a treatment administered to asubject who exhibits signs of pathology for the purpose of diminishingor eliminating those signs.

[0081] “Diagnostic” means identifying the presence or nature of apathologic condition. Diagnostic methods differ in their sensitivity andspecificity. The “sensitivity” of a diagnostic assay is the percentageof diseased individuals who test positive (percent of true positives).The “specificity” of a diagnostic assay is 1 minus the false positiverate, where the false positive rate is defined as the proportion ofthose without the disease who test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis.

[0082] “Prognostic” means predicting the probable development (e.g.,severity) of a pathologic condition.

[0083] “Test amount” refers to an amount of an analyte in a subjectsample, which is then compared to a normal amount of the analyte in asample (e.g., from a healthy individual) such that the relativecomparison of the values provides a reference value for diagnosing adesignated disease. Depending upon the method of detection, the testamount may be a determination of the amount of the analyte, but it isnot necessarily an amount. The test amount may also be a relative value,such as a plus or a minus score, and also includes an amount indicatingthe presence or absence of the analyte in a sample.

[0084] “Normal amount” refers to an amount or a range of an analyte in abiological sample that indicates health or lack of pathology.

[0085] “Diagnostic amount” refers to an amount of an analyte in asubject sample that is consistent with a particular diagnosis for adesignated disease.

[0086] “Prognostic amount” refers to an amount or range of an analyte ina subject sample that is consistent with a particular prognosis for adesignated disease.

[0087] “Plurality” means at least two.

[0088] An “epitope” is portion of a molecule that specifically binds toan antibody or a T cell receptor. An peptide epitope generally comprisesa sequence of at least 6 amino acids from a polypeptide, although longerand shorter peptides can constitute epitopes.

[0089] “MHC Class I molecule” refers to a heterodimer found on thesurface of cells that present processed antigenic peptides to T cells.The molecule comprises an α chain and a β-microglobulin chain. The αchain contains the antigenic peptide binding site in the α1 and α2domains. The α chain also contains a transmembrane portion that can beremoved without eliminating antigen binding.

[0090] “MHC Class II molecule” refers to a heterodimer found on thesurface of cells that present processed antigenic peptide to T cells. Itcomprises an α chain and a β chain. The antigenic peptide binding siteis located in the al domain of the α chain and the β1 domain of the βchain. However, a single α chain or β chain suffices to bind anantigenic peptide. The α chain and β chain also contain transmembraneregions that can be removed without eliminating antigenic peptidebinding function.

[0091] “T cell receptor” refers to a heterodimer found on the surface ofT cells comprising an α chain and a β chain or a γ and a δ chain. T cellreceptors recognize processed antigens associated with MHC molecules.

[0092] II. cDNA Encoding Repro-EN-1.0 and IB1

[0093] We have isolated a cDNA molecule encoding an autoantigenassociated with endometriosis. The autoantigen is called Repro-EN-1.0.The presence of antibodies that specifically bind to an epitope of theRepro-EN-1.0 polypeptide is a highly sensitive and specific diagnosticmarker for endometriosis.

[0094] Polynucleotides encoding full-length Repro-EN-1.0 are useful inrecombinant production Repro-EN-1.0 or immunogenic fragments of it.Fragments of polynucleotides encoding Repro-EN-1.0 are useful as probesto detect Repro-EN-1.0 mRNA from certain cell types suspected to becancerous. Fragments also are useful as primers for amplification ofsequences from Repro-EN-1.0.

[0095] The Repro-EN-1.0 polypeptide and immunogenic fragments of it areuseful as positive controls in diagnostic assays to detect antibodiesthat specifically bind to Repro-EN-1.0 from patient serum samples. Thepolypeptides also are useful as immunogens for eliciting production ofantibodies against epitopes of the protein.

[0096] The nucleotide sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of Repro-EN-1.0 follow:CGGCCGGGCTTCAGGGGCCCAGGCGCCGCTGCTGCCACCGCCATCTAACGCTGCGCCCTG 1|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 60GCCGGCCCGAAGTCCCCGGGTCCGCGGCGACGACGGTGGCGGTAGATTGCGACGCGGGACGAGGCCCGGCGCGCGGATGGTGCCGGTGCGGCTCGGGTGTTGAAACGGGTGTCCCCTCCC 61++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 120CTCCGGGCCGCGCGCCTACCACGGCCACGCCGAGCCCACAACTTTGCCCACAGGGGAGGGCCTCCTCCCCTCCCCCACGCGGTGGTCTCCCCTCCCACCCGGCTCAGGCAGAGCCATGTC 121++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 180GGAGGAGGGGAGGGGGTGCGCCACCAGAGGGGAGGGTGGGCCGAGTCCGTCTCGGTACAG                                                      |M  STCGGGGTGGCTCCTACCCACACCTGTTGTGGGACGTGAGGAAAAGGTTCCTCGGGCTGGA 181++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 240AGCCCCACCGAGGATGGGTGTGGACAACACCCTGCACTCCTTTTCCAAGGAGCCCGACCT  R  G  G  S  Y  P  H  L  L  W  D  V  R  K  R  F  L  G  L  EGGACCCGTCCCGGCTGCGGAGTCGCTACCTGGGAAGAAGAGAATTTATCCAAAGATTAAA 241++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 300CCTGGGCAGGGCCGACGCCTCAGCGATGGACCCTTCTTCTCTTAAATAGGTTTCTAATTT  D  P  S  R  L  R  S  R  Y  L  G  R  R  E  F  I  Q  R  L  KACTTGAAGCAACCCTTAATGTGCATGATGGTTGTGTTAATACAATCTGTTGGAATGACAC 301++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 360TGAACTTCGTTGGGAATTACACGTACTACCAACACAATTATGTTAGACAACCTTACTGTG  L  E  A  T  L  N  V  H  D  G  C  V  N  T  I  C  W  N  D  TTGGAGAATATATTTTATCTGGCTCAGATGACACCAAATTAGTAATTAGTAATCCTTACAG 361++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 420ACCTCTTATATAAAATAGACCGAGTCTACTGTGGTTTAATCATTAATCATTAGGAATGTC  G  E  Y  I  L  S  G  S  D  D  T  K  L  V  I  S  N  P  Y  SCAGAAAGGTTTTGACAACAATTCGTTCAGGGCACCGAGCAAACATATTTAGTGCAAAGTT 421++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 480GTCTTTCCAAAACTGTTGTTAAGCAAGTCCCGTGGCTCGTTTGTATAAATCACGTTTCAA  R  K  V  L  T  T  I  R  S  G  H  R  A  N  I  F  S  A  K  FCTTACCTTGTACAAATGATAAACAGATTGTATCCTGCTCTGGAGATGGAGTAATATTTTA 481++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 540GAATGGAACATGTTTACTATTTGTCTAACATAGGACGAGACCTCTACCTCATTATAAAAT  L  P  C  T  N  D  K  Q  I  V  S  C  S  G  D  G  V  I  F  YTACCAACGTTGAGCAAGATGCAGAAACCAACAGACAATGCCAATTTACGTGTCATTATGG 541++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 600ATGGTTGCAACTCGTTCTACGTCTTTGGTTGTCTGTTACGGTTAAATGCACAGTAATACC  T  N  V  E  Q  D  A  E  T  N  R  Q  C  Q  F  T  C  H  Y  GAACTACTTATGAGATTATGACTGTACCCAATGACCCTTACACTTTTCTCTCTTGTGGTGA 600++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 660TTGATGAATACTCTAATACTGACATGGGTTACTGGGAATGTGAAAAGAGAGAACACCACT  T  T  Y  E  I  M  T  V  P  N  D  P  Y  T  F  L  S  C  G  EAGATGGAACTGTTAGGTGGTTTGATACACGCATCAAAACTAGCTGCACAAAAGAAGATTG 661++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 720TCTACCTTGACAATCCACCAAACTATGTGCGTAGTTTTGATCGACGTGTTTTCTTCTAAC  D  G  T  V  R  W  F  D  T  R  I  K  T  S  C  T  K  E  D  CTAAAGATGATATTTTAATTAACTGTCGACGTGCTGCCACGTCTGTTGCTATTTGCCCACC 721++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 780ATTTCTACTATAAAATTAATTGACAGCTGCACGACGGTGCAGACAACGATAAACGGGTGG  K  D  D  I  L  I  N  C  R  R  A  A  T  S  V  A  I  C  P  PAATACCATATTACCTTGCTGTTGGTTGTTCTGACAGCTCAGTACGAATATATGATCGGCG 781++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 840TTATGGTATAATGGAACGACAACCAACAAGACTGTCGAGTCATGCTTATATACTAGCCGC  I  P  Y  Y  L  A  V  G  C  S  D  S  S  V  R  I  Y  D  R  RAATGCTGGGCACAAGAGCTACAGGGAATTATGCAGGTCGAGGGACTACTGGAATGGTTGC 841++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 900TTACGACCCGTGTTCTCGATGTCCCTTAATACGTCCAGCTCCCTGATGACCTTACCAACG  M  L  G  T  R  A  T  G  N  Y  A  G  R  G  T  T  G  M  V  ACCGTTTTATTCCTTCCCATCTTAATAATAAGTCCTGCAGAGTGACATCTCTGTGTTACAG 901++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 960GGCAAAATAAGGAAGGGTAGAATTATTATTCAGGACGTCTCACTGTAGAGACACAATGTC  R  F  I  P  S  H  L  N  N  K  S  C  R  V  T  S  L  C  Y  STGAAGATGGTCAAGAGATTCTCGTTAGTTACTCTTCAGATTACATATATCTTTTTGACCC 961++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1020ACTTCTACCAGTTCTCTAAGAGCAATCAATGAGAAGTCTAATGTATATAGAAAAACTGGG  E  D  G  Q  E  I  L  V  S  Y  S  S  D  Y  I  Y  L  F  D  PGAAAGATGATACAGCACGAGAACTTAAAACTCCTTCTGCGGAAGAGAGAAGAGAAGAGTT 1021++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1080CTTTCTACTATGTCGTGCTCTTGAATTTTGAGGAAGACGCCTTCTCTCTTCTCTTCTCAA  K  D  D  T  A  R  E  L  K  T  P  S  A  E  E  R  R  E  E  LGCGACAACCACCAGTTAAGCGTTTGAGACTTCGTGGTGATTGGTCAGATACTGGACCCAG 1081++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1040CGCTGTTGGTGGTCAATTCGCAAACTCTGAAGCACCACTAACCAGTCTATGACCTGGGTC  R  Q  P  P  V  K  R  L  R  L  R  G  D  W  S  D  T  G  P  RAGCAAGGCCGGAGAGTGAACGAGAACGAGATGGAGAGCAGAGTCCCAATGTGTCATTGAT 1141++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1200TCGTTCCGGCCTCTCACTTGCTCTTGCTCTACCTCTCGTCTCAGGGTTACACAGTAACTA  A  R  P  E  S  E  R  E  R  D  G  E  Q  S  P  N  V  S  L  MGCAGAGAATGTCTGATATGTTATCAAGATGGTTTGAAGAAGCAAGTGAGGTTGCACAAAG 1201++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1260CGTCTCTTACAGACTATACAATAGTTCTACCAAACTTCTTCGTTCACTCCAACGTGTTTC  Q  R  M  S  D  M  L  S  R  W  F  E  E  A  S  E  V  A  Q  SCAATAGAGGACGAGGAAGATCTCGACCCAGAGGTGGAACAAGTCAATCAGATATTTCAAC 1261++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1320GTTATCTCCTGCTCCTTCTAGAGCTGGGTCTCCACCTTGTTCAGTTAGTCTATAAAGTTG  N  R  G  R  G  R  S  R  P  R  G  G  T  S  Q  S  D  I  S  TTCTTCCTACGGTCCCATCAAGTCCTGATTTGGAAGTGAGTGAAACTGCAATGGAAGTAGA 1321++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1380AGAAGGATGCCAGGGTAGTTCAGGACTAAACCTTCACTCACTTTGACGTTACCTTCATCT  L  P  T  V  P  S  S  P  D  L  E  V  S  E  T  A  M  E  V  DTACTCCAGCTGAACAATTTCTTCAGCCTTCTACATCCTCTACAATGTCAGCTCAGGCTCA 1381++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1440ATGAGGTCGACTTGTTAAAGAAGTCGGAAGATGTAGGAGATGTTACAGTCGAGTCCGAGT  T  P  A  E  Q  F  L  Q  P  S  T  S  S  T  M  S  A  Q  A  HTTCGACATCATCTCCCACAGAAAGCCCTCATTCTACTCCTTTGCTATCTTCTCCAGATAG 1441++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1500AAGCTGTAGTAGAGGGTGTCTTTCGGGAGTAAGATGAGGAAACGATAGAAGAGGTCTATC  S  T  S  S  P  T  E  S  P  H  S  T  P  L  L  S  S  P  D  STGAACAAAGGCAGTCTGTTGAGGCATCTGGACACCACACACATCATCAGTCTGATAACAA 1501++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1560ACTTGTTTCCGTCAGACAACTCCGTAGACCTGTGGTGTGTGTAGTAGTCAGACTATTGTT  E  Q  R  Q  S  V  E  A  S  G  H  H  T  H  H  Q  S  D  N  NTAATGAAAAGCTGAGCCCCAAACCAGGGACAGGTGAACCAGTTTTAAGTTTGCACTACAG 1561++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1620ATTACTTTTCGACTCGGGGTTTGGTCCCTGTCCACTTGGTCAAAATTCAAACGTGATGTC  N  E  K  L  S  P  K  P  G  T  G  E  P  V  L  S  L  H  Y  SCACAGAAGGAACAACTACAAGCACAATAAAACTGAACTTTACAGATGAATGGAGCAGTAT 1621++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1680GTGTCTTCCTTGTTGATGTTCGTGTTATTTTGACTTGAAATGTCTACTTACCTCGTCATA  T  E  G  T  T  T  S  T  I  K  L  N  F  T  D  E  W  S  S  IAGCATCAAGTTCTAGAGGAATTGGGAGCCATTGCAAATCTGAGGGTCAGGAGGAATCTTT 1681++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1740TCGTAGTTCAAGATCTCCTTAACCCTCGGTAACGTTTAGACTCCCAGTCCTCCTTAGAAA  A  S  S  S  R  G  I  G  S  H  C  K  S  E  G  Q  E  E  S  FCGTCCCACAGAGCTCAGTGCAACCACCAGAAGGAGACAGTGAAACAAAAGCTCCTGAAGA 1741++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1800GCAGGGTGTCTCGAGTCACGTTGGTGGTCTTCCTCTGTCACTTTGTTTTCGAGGACTTCT  V  P  Q  S  S  V  Q  P  P  E  G  D  S  E  T  K  A  P  E  EATCATCAGAGGATGTGACAAAATATCAGGAAGGAGTATCTGCAGAAAACCCAGTTGAGAA 1801++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1860TAGTAGTCTCCTACACTGTTTTATAGTCCTTCCTCATAGACGTCTTTTGGGTCAACTCTT  S  S  E  D  V  T  K  Y  Q  E  G  V  S  A  E  N  P  V  E  NCCATATCAATATAACACAATCAGATAAGTTCACAGCCAAGCCATTGGATTCCAACTCAGG 1861++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1920GGTATAGTTATATTGTGTTAGTCTATTCAAGTGTCGGTTCGGTAACCTAAGGTTGAGTCC  H  I  N  I  T  Q  S  D  K  F  T  A  K  P  L  D  S  N  S  GAGAAAGAAATGACCTCAATCTTGATCGCTCTTGTGGGGTTCCAGAAGAATCTGCTTCATC 1921++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1980TCTTTCTTTACTGGAGTTAGAACTAGCGAGAACACCCCAAGGTCTTCTTAGACGAAGTAG  E  R  N  D  L  N  L  D  R  S  C  G  V  P  E  E  S  A  S  STGAAAAAGCCAAGGAACCAGAAACTTCAGATCAGACTAGCACTGAGAGTGCTACCAATGA 1981++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2040ACTTTTTCGGTTCCTTGGTCTTTGAAGTCTAGTCTGATCGTGACTCTCACGATGGTTACT  E  K  A  K  E  P  E  T  S  D  Q  T  S  T  E  S  A  T  N  EAAATAACACCAATCCTGAGCCTCAGTTCCAAACAGAAGCCACTGGGCCTTCAGCTCATGA 2041++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2100TTTATTGTGGTTAGGACTCGGAGTCAAGGTTTGTCTTCGGTGACCCGGAAGTCGAGTACT  N  N  T  N  P  E  P  Q  F  Q  T  E  A  T  G  P  S  A  H  EAGAAACATCCACCAGGGACTCTGCTCTTCAGGACACAGATGACAGTGATGATGACCCAGT 2101++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2160TCTTTGTAGGTGGTCCCTGAGACGAGAAGTCCTGTGTCTACTGTCACTACTACTGGGTCA  E  T  S  T  R  D  S  A  L  Q  D  T  D  D  S  D  D  D  P  VCCTGATCCCAGGTGCAAGGTATCGAGCAGGACCTGGTGATAGACGCTCTGCTGTTGCCCG 21-61++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2220GGACTAGGGTCCACGTTCCATAGCTCGTCCTGGACCACTATCTGCGAGACGACAACGGGC  L  I  P  G  A  R  Y  R  A  G  P  G  D  R  R  S  A  V  A  RTATTCAGGAGTTCTTCAGACGGAGAAAAGAAAGGAAAGAAATGGAAGAATTGGATACTTT 2221++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2280ATAAGTCCTCAAGAAGTCTGCCTCTTTTCTTTCCTTTCTTTACCTTCTTAACCTATGAAA  I  Q  E  F  F  R  R  R  K  E  R  K  E  M  E  E  L  D  T  LGAACATTAGAAGGCCGCTAGTAAAAATGGTTTATAAAGGCCATCGCAACTCCAGGACAAT 2281++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2340CTTGTAATCTTCCGGCGATCATTTTTACCAAATATTTCCGGTAGCGTTGAGGTCCTGTTA  N  I  R  R  P  L  V  K  M  V  Y  K  G  H  R  N  S  R  T  MGATAAAAGAAGCCAATTTCTGGGGTGCTAACTTTGTAATGACTGGTTCTGAGTGTGGCCA 2341++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2400CTATTTTCTTCGGTTAAAGACCCCACGATTGAAACATTACTGACCAAGACTCACACCGGT  I  K  E  A  N  F  W  G  A  N  F  V  M  T  G  S  E  C  G  HCATTTTCATCTGGGATCGGCACACTGCTGAGCATTTGATGCTTCTGGAAGCTGATAATCA 2401++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2460GTAAAAGTAGACCCTAGCCGTGTGACGACTCGTAAACTACGAAGACCTTCGACTATTAGT  I  F  I  W  D  R  H  T  A  E  H  L  M  L  L  E  A  D  N  HTGTGGTAAACTGCCTGCAGCCACATCCGTTTGACCCAATTTTAGCCTCATCTGGCATAGA 2461++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2520ACACCATTTGACGGACGTCGGTGTAGGCAAACTGGGTTAAAATCGGAGTAGACCGTATCT  V  V  N  C  L  Q  P  H  P  F  D  P  I  L  A  S  S  G  I  DTTATGACATAAAGATCTGGTCACCATTAGAAGAGTCAAGGATTTTTAACCGAAAACTTGC 2521++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2580AATACTGTATTTCTAGACCAGTGGTAATCTTCTCAGTTCCTAAAAATTGGCTTTTGAACG  Y  D  I  K  I  W  S  P  L  E  E  S  R  I  F  N  R  K  L  ATGATGAAGTTATAACTCGAAACGAACTCATGCTGGAAGAAACTAGAAACACCATTACAGT 2581++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2640ACTACTTCAATATTGAGCTTTGCTTGAGTACGACCTTCTTTGATCTTTGTGGTAATGTCA  D  E  V  I  T  R  N  E  L  M  L  E  E  T  R  N  T  I  T  VTCCAGCCTCTTTCATGTTGAGGATGTTGGCTTCACTTAATCATATCCGAGCTGACCGGTT 2641++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2700AGGTCGGAGAAAGTACAACTCCTACAACCGAAGTGAATTAGTATAGGCTCGACTGGCCAA  P  A  S  F  M  L  R  M  L  A  S  L  N  H  I  R  A  D  R  LGGAGGGTGACAGATCAGAAGGCTCTGGTCAAGAGAATGAAAATGAGGATGAGGAATAATA 2701++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2760CCTCCCACTGTCTAGTCTTCCGAGACCAGTTCTCTTACTTTTACTCCTACTCCTTATTAT  E  G  D  R  S  E  G  S  G  Q  E  N  E  N  E  D  E  E  . |AACTCTTTTTGGCAAGCACTTAAATGTTCTGAAATTTGTATAAGACATTTATTATTTTTT 2761++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2820TTGAGAAAAACCGTTCGTGAATTTACAAGACTTTAAACATATTCTGTAAATAATAAAAAATTTCTTTACAGAGATTTAGTGCAATTTTAAGGTTATGGTTTTTGGAGTTTTTCCCTTTTT 2821++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2880AAAGAAATGTCTCTAAATCACGTTAAAATTCCAATACCAAAAACCTCAAAAAGGGAAAAATTGGGATAACCTAACATTGGTTTGGAATGATTGTGTGCATGAATTTGGGAGATTGTATAA 2881++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2940AACCCTATTGGATTGTAACCAAACCTTACTAACACACGTACTTAAACCCTCTAACATATTAACAAAACTAGCAGAATGTTTTTAAAACTTTTTGCCGTGTATGAGGAGTGCTAGAAAATG 2941++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3000TTGTTTTGATCGTCTTACAAAAATTTTGAAAAACGGCACATACTCCTCACGATCTTTTACCAAAGTGCAATATTTTCCCTAACCTTCAAATGTGGGAGCTTGGATCAATGTTGAAGAATA 3001++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3060GTTTCACGTTATAAAAGGGATTGGAAGTTTACACCCTCGAACCTAGTTACAACTTCTTATATTTTCATCATAGTGAAAATGTTGGTTCAAATAAATTTCTACACTTGCCATTTGCATGTT 3061++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3120TAAAAGTAGTATCACTTTTACAACCAAGTTTATTTAAAGATGTGAACGGTAAACGTACAATGTTGCTTTCTAATTAAAGAAACTGGTTGTTTTAAGATACCCTGAAAAAAAAAAAAAAAA 3121++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3180ACAACGAAAGATTAATTTCTTTGACCAACAAAATTCTATGGGACTTTTTTTTTTTTTTTT AAAAAAAAA3181 ++++|++→ 3189 TTTTTTTTT

[0097] This 3164-base nucleotide sequence contains an open reading frameof 2580 nucleotides encoding Repro-EN-1.0 from nucleotide 176 tonucleotide 2755. The deduced amino acid sequence of Repro-EN-1.0 has 860amino acids. Repro-EN-1.0 has a calculated molecular mass of 96.4 kD anda pI of 5.08.

[0098] The Repro-EN-1.0 gene encodes a 3.4 kb mRNA. This mRNA isexpressed primarily in skeletal muscle, heart and testis, and to alesser extent in other tissues. However, it is not detected in lung orperipheral blood mononuclear cells (PBMC). Expression of Repro-EN-1.0 isup-regulated in breast and uterine carcinomas relative to their normalcounterparts. It is highly expressed in both normal fallopian tube andfallopian tube carcinoma. It is expressed in low levels in normal ovaryand ovarian carcinoma. Expression of the mRNA is lower in endometrialcarcinoma cell lines than in prostate adenocarcinoma cell lines.

[0099] Analysis of the deduced amino acid sequence of Repro-EN-1.0 showsno significant sequence identity with any other protein.

[0100] There is an alternately spliced variant that was isolated form ahuman heart cDNA library. This variant is called IB1 and is useful inthe same ways as the nucleotide sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:2) of Repro-EN-1.0. The IB1 sequence wasisolated from a heart cDNA library by screening with a nucleic acidprobe obtained from the Repro-EN-1.0 sequence. The IB1 sequence isdifferent from the Repro-EN-1.0 sequence in that it contains anadditional 231 bp exon inserted into the cDNA sequence at position 1555.Therefore the IB1 sequence has similar properties, but is slightlylarger.

[0101] The nucleotide sequence (SEQ ID NO:3) and the deduced amino acidsequence (SEQ ID NO:4) of IB1 follow:CGGCCGGGCTTCAGGGGCCCAGGCGCCGCTGCTGCCACCGCCATCTAACGCTGCGCCCTG 1|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 60GCCGGCCCGAAGTCCCCGGGTCCGCGGCGACGACGGTGGCGGTAGATTGCGACGCGGGACGAGGCCCGGCGCGCGGATGGTGCCGGTGCGGCTCGGGTGTTGAAACGGGTGTCCCCTCCC 61++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 120CTCCGGGCCGCGCGCCTACCACGGCCACGCCGAGCCCACAACTTTGCCCACAGGGGAGGGCCTCCTCCCCTCCCCCACGCGGTGGTCTCCCCTCCCACCCGGCTCAGGCAGAGCCATGTC 121++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 180GGAGGAGGGGAGGGGGTGCGCCACCAGAGGGGAGGGTGGGCCGAGTCCGTCTCGGTACAG                                                       |M  STCGGGGTGGCTCCTACCCACACCTGTTGTGGGACGTGAGGAAAAGGTTCCTCGGGCTGGA 181++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 240AGCCCCACCGAGGATGGGTGTGGACAACACCCTGCACTCCTTTTCCAAGGAGCCCGACCT  R  G  G  S  Y  P  H  L  L  W  D  V  R  K  R  F  L  G  L  EGGACCCGTCCCGGCTGCGGAGTCGCTACCTGGGAAGAAGAGAATTTATCCAAAGATTAAA 241++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 300CCTGGGCAGGGCCGACGCCTCAGCGATGGACCCTTCTTCTCTTAAATAGGTTTCTAATTT  D  P  S  R  L  R  S  R  Y  L  G  R  R  E  F  I  Q  R  L  KACTTGAAGCAACCCTTAATGTGCATGATGGTTGTGTTAATACAATCTGTTGGAATGACAC 301++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 360TGAACTTCGTTGGGAATTACACGTACTACCAACACAATTATGTTAGACAACCTTACTGTG  L  E  A  T  L  N  V  H  D  G  C  V  N  T  I  C  W  N  D  TTGGAGAATATATTTATCTGGCTCAGATGACACCAAATTAGTAATTAGTAATCCTTACAG 361++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 420ACCTCTTATATAAAATAGACCGAGTCTACTGTGGTTTAATCATTAATCATTAGGAATGTC  G  E  Y  I  L  S  G  S  D  D  T  K  L  V  I  S  N  P  Y  SCAGAAAGGTTTTGACAACAATTCGTTCAGGGCACCGAGCAAACATATTTAGTGCAAAGTT 421++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 480GTCTTTCCAAAACTGTTGTTAAGCAAGTCCCGTGGCTCGTTTCTATAAATCACGTTTCAA  R  K  V  L  T  T  I  R  S  G  H  R  A  N  I  F  S  A  K  FCTTACCTTGTACAAATGATAAACAGATTGTATCCTGCTCTGGAGATGGAGTAATATTTTA 481++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 540GAATGGAACATGTTTACTATTTGTCTAACATAGGACGAGACCTCTACCTCATTATAAAAT  L  P  C  T  N  D  K  Q  I  V  S  C  S  G  D  G  V  I  F  YTACCAACGTTGAGCAAGATGCAGAAACCAACAGACAATGCCAATTTACGTGTCATTATGG 541++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 600ATGGTTGCAACTCGTTCTACGTCTTTGGTTGTCTGTTACGGTTAAATGCACAGTAATACC  T  N  V  E  Q  D  A  E  T  N  R  Q  C  Q  F  T  C  H  Y  GAACTACTTATGAGATTATGACTGTACCCAATGACCCTTACACTTTTCTCTCTTGTGGTGA 601++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 660TTGATGAATACTCTAATACTGACATGGGTTACTGGGAATGTGAAAAGAGAGAACACCACT  T  T  Y  E  I  M  T  V  P  N  D  P  Y  T  F  L  S  C  G  EAGATGGAACTGTTAGGTGGTTTGATACACGCATCAAAACTAGCTGCACAAAAGAAGATTG 661++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 720TCTACCTTGACAATCCACCAAACTATGTGCGTAGTTTTGATCGACGTGTTTTCTTCTAAC  D  G  T  V  R  W  F  D  T  R  I  K  T  S  C  T  K  E  D  CTAAAGATGATATTTTAATTAACTGTCGACGTGCTGCCACGTCTGTTGCTATTTGCCCACC 721++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 780ATTTCTACTATAAAATTAATTGACAGCTGCACGACGGTGCAGACAACGATAAACGGGTGG  K  D  D  I  L  I  N  C  R  R  A  A  T  S  V  A  I  C  P  PAATACCATATTACCTTGCTGTTGGTTGTTCTGACAGCTCAGTACGAATATATGATCGGCG 781++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 840TTATGGTATAATGGAACGACAACCAACAAGACTGTCGAGTCATGCTTATATACTAGCCGC  I  P  Y  Y  L  A  V  G  C  S  D  S  S  V  R  I  Y  D  R  RAATGCTGGGCACAAGAGCTACAGGGAATTATGCAGGTCGAGGGACTACTGGAATGGTTGC 841++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 900TTACGACCCGTGTTCTCGATGTCCCTTAATACGTCCAGCTCCCTGATGACCTTACCAACG  M  L  G  T  R  A  T  G  N  Y  A  G  R  G  T  T  G  M  V  ACCGTTTTATTCCTTCCCATCTTAATAATAAGTCCTGCAGAGTGACATCTCTGTGTTACAG 901++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 960GGCAAAATAAGGAAGGGTAGAATTATTATTCAGGACGTCTCACTGTAGAGACACAATGTC  R  F  I  P  S  H  L  N  N  K  S  C  R  V  T  S  L  C  Y  STGAAGATGGTCAAGAGATTCTCGTTAGTTACTCTTCAGATTACATATATCTTTTTGACCC 961++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1020ACTTCTACCAGTTCTCTAAGAGCAATCAATGAGAAGTCTAATGTATATAGAAAAACTGGG  E  D  G  Q  E  I  L  V  S  Y  S  S  D  Y  I  Y  L  F  D  PGAAAGATGATACAGCACGAGAACTTAAAACTCCTTCTGCGGAAGAGAGAAGAGAAGAGTT 1021++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1080CTTTCTACTATGTCGTGCTCTTGAATTTTGAGGAAGACGCCTTCTCTCTTCTCTTCTCAA  K  D  D  T  A  R  E  L  K  T  P  S  A  E  E  R  R  E  E  LGCGACAACCACCAGTTAAGCGTTTGAGACTTCGTGGTGATTGGTCAGATACTGGACCCAG 1081++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1140CGCTGTTGGTGGTCAATTCGCAAACTCTGAAGCACCACTAACCAGTCTATGACCTGGGTC  R  Q  P  P  V  K  R  L  R  L  R  G  D  W  S  D  T  G  P  RAGCAAGGCCGGAGAGTGAACGAGAACGAGATGGAGAGCAGAGTCCCAATGTGTCATTGAT 1141++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1200TCGTTCCGGCCTCTCACTTGCTCTTGCTCTACCTCTCGTCTCAGGGTTACACAGTAACTA  A  R  P  E  S  E  R  E  R  D  G  E  Q  S  P  N  V  S  L  MGCAGAGAATGTCTGATATGTTATCAAGATGGTTTGAAGAAGCAAGTGAGGTTGCACAAAG 1201++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1260CGTCTCTTACAGACTATACAATAGTTCTACCAAACTTCTTCGTTCACTCCAACGTGTTTC  Q  R  M  S  D  M  L  S  R  W  F  E  E  A  S  E  V  A  Q  SCAATAGAGGACGAGGAAGATCTCGACCCAGAGGTGGAACAAGTCAATCAGATATTTCAAC 1261++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1320GTTATCTCCTGCTCCTTCTAGAGCTGGGTCTCCACCTTGTTCAGTTAGTCTATAAAGTTG  N  R  G  R  G  R  S  R  P  R  G  G  T  S  Q  S  D  I  S  TTCTTCCTACGGTCCCATCAAGTCCTGATTTGGAAGTGAGTGAAACTGCAATGGAAGTAGA 1321++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1380AGAAGGATGCCAGGGTAGTTCAGGACTAAACCTTCACTCACTTTGACGTTACCTTCATCT  L  P  T  V  P  S  S  P  D  L  E  V  S  E  T  A  M  E  V  DTACTCCAGCTGAACAATTTCTTCAGCCTTCTACATCCTCTACAATGTCAGCTCAGGCTCA 1381++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1440ATGAGGTCGACTTGTTAAAGAAGTCGGAAGATGTAGGAGATGTTACAGTCGAGTCCGAGT  T  P  A  E  Q  F  L  Q  P  S  T  S  S  T  M  S  A  Q  A  HTTCGACATCATCTCCCACAGAAAGCCCTCATTCTACTCCTTTGCTATCTTCTCCAGATAG 1441++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1500AAGCTGTAGTAGAGGGTGTCTTTCGGGAGTAAGATGAGGAAACGATAGAAGAGGTCTATC  S  T  S  S  P  T  E  S  P  H  S  T  P  L  L  S  S  P  D  STGAACAAAGGCAGTCTGTTGAGGCATCTGGACACCACACACATCATCAGTCTGAATTTTT 1501++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1560ACTTGTTTCCGTCAGACAACTCCGTAGACCTGTGGTGTGTGTAGTAGTCAGACTTAAAAA  E  Q  R  Q  S  V  E  A  S  G  H  H  T  H  H  Q  S  E  F  L                                                      {overscore(|------)} AAGGGGGCCTGAGATAGCTTTGCTTCGTAAGCGCCTGCAACAACTGAGGCTTAAGAAGGC1561 ++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1620TTCCCCCGGACTCTATCGAAACGAAGCATTCGCGGACGTTGTTGACTCCGAATTCTTCCG  R  G  P  E  I  A  L  L  R  K  R  L  Q  Q  L  R  L  K  K  A {overscore(                         231 bp insert                      )}TGAGCAGCAGAGGCAGCAAGAGCTAGCTGCACATACCCAGCAACAGCCTTCCACTTCTGA 1621++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1680ACTCGTCGTCTCCGTCGTTCTCGATCGACGTGTATGGTCGTTGTCGGAAGGTGAAGACT  E  Q  Q  R  Q  Q  E  L  A  A  H  T  Q  Q  Q  P  S  T  S  D {overscore(                         231 bp insert                      )}TCAGTCTTCTCATGAGGGCTCTTCACAGGACCCTCATGCTTCAGATTCTCCTTCTTCTGT 1681++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1680AGTCAGAAGAGTACTCCCGAGAAGTGTCCTGGGAGTACGAAGTCTAAGAGGAAGAAGACA  Q  S  S  H  E  G  S  S  Q  D  P  H  A  S  D  S  P  S  S  V {overscore(                         231 bp insert                      )}GGTTAACAAACAGCTCGGATCCATGTCACTTGACGAGCAACAGGATAACAATAATGAAAA 1741++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1800CCAATTGTTTGTCGAGCCTAGGTACAGTGAACTGCTCGTTGTCCTATTGTTATTACTTTT  V  N  K  Q  L  G  S  M  S  L  D  E  Q  Q  D  N  N  N  E  K {overscore(                 231 bp insert              )} _(/) ^(\)GCTGAGCCCCAAACCAGGGACAGGTGAACCAGTTTTAAGTTTGCACTACAGCACAGAAGG 1801++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1860CGACTCGGGGTTTGGTCCCTGTCCACTTGGTCAAAATTCAAACGTGATGTCGTGTCTTCC  L  S  P  K  P  G  T  G  E  P  V  L  S  L  H  Y  S  T  E  GAACAACTACAAGCACAATAAAACTGAACTTTACAGATGAATGGAGCAGTATAGCATCAAG 1861++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1920TTGTTGATGTTCGTGTTATTTTGACTTGAAATGTCTACTTACCTCGTCATATCGTAGTTC  T  T  T  S  T  I  K  L  N  F  T  D  E  W  S  S  I  A  S  STTCTAGAGGAATTGGGAGCCATTGCAAATCTGAGGGTCAGGAGGAATCTTTCGTCCCACA 1921++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 1980AAGATCTCCTTAACCCTCGGTAACGTTTAGACTCCCAGTCCTCCTTAGAAAGCAGGGTGT  S  R  G  I  G  S  H  C  K  S  E  G  Q  E  E  S  F  V  P  QGAGCTCAGTGCAACCACCAGAAGGAGACAGTGAAACAAAAGCTCCTGAAGAATCATCAGA 1981++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2040CTCGAGTCACGTTGGTGGTCTTCCTCTGTCACTTTGTTTTCGAGGACTTCTTAGTAGTCT  S  S  V  Q  P  P  E  G  D  S  E  T  K  A  P  E  E  S  S  EGGATGTGACAAAATATCAGGAAGGAGTATCTGCAGAAAACCCAGTTGAGAACCATATCAA 2041++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2100CCTACACTGTTTTATAGTCCTTCCTCATAGACGTCTTTTGGGTCAACTCTTGGTATAGTT  D  V  T  K  Y  Q  E  G  V  S  A  E  N  P  V  E  N  H  I  NTATAACACAATCAGATAAGTTCACAGCCAAGCCATTGGATTCCAACTCAGGAGAAAGAAA 2101++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2160ATATTGTGTTAGTCTATTCAAGTGTCGGTTCGGTAACCTAAGGTTGAGTCCTCTTTCTTT  I  T  Q  S  D  K  F  T  A  K  P  L  D  S  N  S  G  E  R  NTGACCTCAATCTTGATCGCTCTTGTGGGGTTCCAGAAGAATCTGCTTCATCTGAAAAAGC 2161++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2220ACTGGAGTTAGAACTAGCGAGAACACCCCAAGGTCTTCTTAGACGAAGTAGACTTTTTCG  D  L  N  L  D  R  S  C  G  V  P  E  E  S  A  S  S  E  K  ACAAGGAACCAGAAACTTCAGATCAGACTAGCACTGAGAGTGCTACCAATGAAAATAACAC 2221++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2280GTTCCTTGGTCTTTGAAGTCTAGTCTGATCGTGACTCTCACGATGGTTACTTTTATTGTG  K  E  P  E  T  S  D  Q  T  S  T  E  S  A  T  N  E  N  N  TCAATCCTGAGCCTCAGTTCCAAACAGAAGCCACTGGGCCTTCAGCTCATGAAGAAACATC 2281++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2340GTTAGGACTCGGAGTCAAGGTTTGTCTTCGGTGACCCGGAAGTCGAGTACTTCTTTGTAG  N  P  E  P  Q  F  Q  T  E  A  T  G  P  S  A  H  E  E  T  SCACCAGGGACTCTGCTCTTCAGGACACAGATGACAGTGATGATGACCCAGTCCTGATCCC 2341++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2400GTGGTCCCTGAGACGAGAAGTCCTGTGTCTACTGTCACTACTACTGGGTCAGGACTAGGG  T  R  D  S  A  L  Q  D  T  D  D  S  D  D  D  P  V  L  I  PAGGTGCAAGGTATCGAGCAGGACCTGGTGATAGACGCTCTGCTGTTGCCCGTATTCAGGA 2401++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2460TCCACGTTCCATAGCTCGTCCTGGACCACTATCTGCGAGACGACAACGGGCATAAGTCCT  G  A  R  Y  R  A  G  P  G  D  R  R  S  A  V  A  R  I  Q  EGTTCTTCAGACGGAGAAAAGAAAGGAAAGAAATGGAAGAATTGGATACTTTGAACATTAG 2461++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2520CAAGAAGTCTGCCTCTTTTCTTTCCTTTCTTTACCTTCTTAACCTATGAAACTTGTAATC  F  F  R  R  R  K  E  R  K  E  M  E  E  L  D  T  L  N  I  RAAGGCCGCTAGTAAAAATGGTTTATAAAGGCCATCGCAACTCCAGGACAATGATAAAAGA 2521++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2580TTCCGGCGATCATTTTTACCAAATATTTCCGGTAGCGTTGAGGTCCTGTTACTATTTTCT  R  P  L  V  K  M  V  Y  K  G  H  R  N  S  R  T  M  I  K  EAGCCAATTTCTGGGGTGCTAACTTTGTAATGACTGGTTCTGAGTGTGGCCACATTTTCAT 2581++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2640TCGGTTAAAGACCCCACGATTGAAACATTACTGACCAAGACTCACACCGGTGTAAAAGTA  A  N  F  W  G  A  N  F  V  M  T  G  S  E  C  G  H  I  F  ICTGGGATCGGCACACTGCTGAGCATTTGATGCTTCTGGAAGCTGATAATCATGTGGTAAA 2641++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2700GACCCTAGCCGTGTGACGACTCGTAAACTACGAAGACCTTCGACTATTAGTACACCATTT  W  D  R  H  T  A  E  H  L  M  L  L  E  A  D  N  H  V  V  NCTGCCTGCAGCCACATCCGTTTGACCCAATTTTAGCCTCATCTGGCATAGATTATGACAT 2701++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2760GACGGACGTCGGTGTAGGCAAACTGGGTTAAAATCGGAGTAGACCGTATCTAATACTGTA  C  L  Q  P  H  P  F  D  P  I  L  A  S  S  G  I  D  Y  D  IAAAGATCTGGTCACCATTAGAAGAGTCAAGGATTTTTAACCGAAAACTTGCTGATGAAGT 2761++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2820TTTCTAGACCAGTGGTAATCTTCTCAGTTCCTAAAAATTGGCTTTTGAACGACTACTTCA  K  I  W  S  P  L  E  E  S  R  I  F  N  R  K  L  A  D  E  VTATAACTCGAAACGAACTCATGCTGGAAGAAACTAGAAACACCATTACAGTTCCAGCCTC 2821++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2880ATATTGAGCTTTGCTTGAGTACGACCTTCTTTGATCTTTGTGGTAATGTCAAGGTCGGAG  I  T  R  N  E  L  M  L  E  E  T  R  N  T  I  T  V  P  A  STTTCATGTTGAGGATGTTGGCTTCACTTAATCATATCCGAGCTGACCGGTTGGAGGGTGA 2881++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 2940AAAGTACAACTCCTACAACCGAAGTGAATTAGTATAGGCTCGACTGGCCAACCTCCCACT  F  M  L  R  M  L  A  S  L  N  H  I  R  A  D  R  L  E  G  DCAGATCAGAAGGCTCTGGTCAAGAGAATGAAAATGAGGATGAGGAATAATAAACTCTTTT 2941++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3000GTCTAGTCTTCCGAGACCAGTTCTCTTACTTTTACTCCTACTCCTTATTATTTGAGAAAA  R  S  E  G  S  G  Q  E  N  E  N  E  D  E  E  . TGGCAAGCACTTAAATGTTCTGAAATTTGTATAAGACATTTATTATTTTTTTTTCTTTAC 3001++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3060ACCGTTCGTGAATTTACAAGACTTTAAACATATTCTGTAAATAATAAAAAAAAAGAAATGAGAGATTTAGTGCAATTTTAAGGTTATGGTTTTTGGAGTTTTTCCCTTTTTTTGGGATAA 3061++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3120TCTCTAAATCACGTTAAAATTCCAATACCAAAAACCTCAAAAAGGGAAAAAAACCCTATTCCTAACATTGGTTTGGAATGATTGTGTGCATGAATTTGGGAGATTGTATAAAACAAAACT 3121++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3180GGATTGTAACCAAACCTTACTAACACACGTACTTAAACCCTCTAACATATTTTGTTTTGAAGCAGAATGTTTTTAAAACTTTTTGCCGTGTATGAGGAGTGCTAGAAAATGCAAAGTGCA 3181++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3240TCGTCTTACAAAAATTTTGAAAAACGGCACATACTCCTCACGATCTTTTACGTTTCACGTATATTTTCCCTAACCTTCAAATGTGGGAGCTTGGATCAATGTTGAAGAATAATTTTCATC 3241++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3300TATAAAAGGGATTGGAAGTTTACACCCTCGAACCTAGTTACAACTTCTTATTAAAAGTAGATAGTGAAAATGTTGGTTCAAATAAATTTCTACACTTGCCATTTGCATGTTTGTTGCTTT 3301++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++| 3360TATCACTTTTACAACCAAGTTTATTTAAAGATGTGAACGGTAAACGTACAAACAACGAAACTAATTAAAGAAACTGGTTGTTTTAAGATACCCTGAAAAAAAAAAAAAAAAAAAAAAAAA 3361++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++|++++→ 3420GATTAATTTCTTTGACCAACAAAATTCTATGGGACTTTTTTTTTTTTTTTTTTTTTTTTT

[0102] IB1 Alternately Spliced Variant From Heart Library:

[0103] IB1 cDNA is 3,395 base pairs

[0104] IB1 open reading frame is 2,811 nucleotides

[0105] IB1 open reading frame maps to nucleotides 176 to 2986

[0106] Deduced amino acid sequence of IB1 has 937 amino acids

[0107] Calculated moecular mass of IB1 protein is 104,969 Daltons (105kD)

[0108] Calculated pI of IB1 protein is 5.17

[0109] Analysis of the amino acid sequence identified several amino acidmotifs that will be apparent to those skilled in the art including a myb1 DNA binding domain, a WD 40 site, an RGD cell-attachment sequence, anN-myristolylation site and several phosphorylation and glycosylationsites. Analysis also shows that the protein is largely hydrophilic. Thisimplies that most of the amino acid sequence is exposed to the immunesystem and can be recognized as epitopes.

[0110] Analysis of expressed sequence tags (ESTs) from a public database(Genbank) identified many overlapping ESTs that, together, covered mostof the Repro-EN-1.0 cDNA sequence.

[0111] III. Repro-EN-1.0 and IB1 Nucleic Acids

[0112] This invention provides recombinant polynucleotides comprising anucleotide sequence encoding Repro-EN-1.0 and IB1 proteins, Repro-EN-1.0and IB1 analogs or fragments of these polypeptides, as described herein.Repro-EN-1.0 or IB1 analogs include 1) immunogenic fragments ofRepro-EN-1.0 or IB1; 2) homologs of Repro-EN-1.0 or IB1 from othermammals (especially primates); 3) fragments of Repro-EN-1.0 or IB1comprising at least 5 consecutive amino acids from the sequence ofRepro-EN-1.0 or IB1; 4) non-naturally occurring polypeptides whosesequences are substantially identical to Repro-EN-1.0; and 5) fusionproteins comprising a Repro-EN-1.0 or IB1 or a Repro-EN-1.0 or IB1analog fused to a second polypeptide moiety. The polynucleotides areuseful for expressing the mRNA or polypeptides they encode and in thepreparation of probes or primers, among other things.

[0113] In one embodiment, the recombinant polynucleotide moleculecomprises a nucleotide sequence encoding a sequence of at least 5 aminoacids selected from the amino acid sequence of Repro-EN-1.0 (SEQ IDNO:2) or IB1 (SEQ ID NO:4). The nucleotide sequence can encode asequence of at least 25 amino acids, at least 100 amino acids or atleast 200 amino acids from SEQ ID NO:2 or SEQ ID NO:4. In oneembodiment, the nucleotide sequence encodes an immunogenic analog. Onesuch immunogenic analog is a polypeptide comprising an epitope thatbinds specifically to an antibody from serum from a subject diagnosedwith endometriosis. In another embodiment, the nucleotide sequenceencodes full-length native Repro-EN-1.0 or IB1 polypeptide.

[0114] The nucleotide sequence can be identical to a sequence fromRepro-EN-1.0 cDNA or its complement or IB1 cDNA or its complement, orcan include degenerate codons. In one embodiment of a nucleotidesequence encoding full-length Repro-EN-1.0 or IB1, the sequence isidentical to the coding sequence of Repro-EN-1.0 of SEQ ID NO:1 or IB1of (SEQ ID NO:3). In another embodiment, the nucleotide sequence encodesa Repro-EN-1.0 or IB1 analog whose amino acid sequence is substantiallyidentical to the amino acid sequence of Repro-EN-1.0 polypeptide (SEQ IDNO:2) or IB1 polypeptide (SEQ ID NO:4).

[0115] In another embodiment, the polynucleotide encodes a fusionprotein between Repro-EN-1.0 or IB1 polypeptide or Repro-EN-1.0 or IB1analog amino acid sequences and a second amino acid sequence. The secondamino acid sequence can be, for example, a detectable label such as afluorescent protein, enzyme marker of protein from a two-hybrid system.

[0116] The polynucleotides of the present invention are cloned oramplified by in vitro methods, such as the polymerase chain reaction(PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (3SR) and the Qβ replicase amplification system (QB). Forexample, a polynucleotide encoding the protein can be isolated bypolymerase chain reaction of cDNA from a human endometrial carcinomacell line using primers based on the DNA sequence of Repro-EN-1.0 of SEQID NO:1. One pair of primers useful for amplifying Repro-EN-1.0 DNA,including allelic variants, is:

[0117] Upstream sense: 5′-caggacacagatgacagtgat-3′ (SEQ ID NO:5)

[0118] Downstream antisense: 5′-agagccttctgatctgtcac-3′ (SEQ ID NO:6).

[0119] A wide variety of cloning and in vitro amplificationmethodologies are well-known to persons of skill. PCR methods aredescribed in, for example, U.S. Pat. No. 4,683,195; Mullis et al. (1987)Cold Spring Harbor Symp. Quant. Biol. 51:263; and Erlich, ed., PCRTechnology, (Stockton Press, NY, 1989). One useful format is real timePCR. See, e.g., Luch et al. (1997) J. Molec. Endocrinol. 18:77-85 andArold et al., (1997) Proc. Nat'l Acad. Sci., USA 94:2438-43.Polynucleotides also can be isolated by screening genomic or cDNAlibraries with probes selected from the sequences of SEQ ID NO:1 understringent hybridization conditions.

[0120] Mutant versions of the proteins can be made by site-specificmutagenesis of other polynucleotides encoding the proteins, or by randommutagenesis caused by increasing the error rate of PCR of the originalpolynucleotide with 0.1 mM MnCl2 and unbalanced nucleotideconcentrations.

[0121] This invention also provides expression vectors, e.g.,recombinant polynucleotide molecules comprising expression controlsequences operatively linked to a nucleotide sequence encoding thetarget polypeptide. Expression vectors can be adapted for function inprokaryotes or eukaryotes by inclusion of appropriate promoters,replication sequences, markers, etc. for transcription and translationof mRNA. The construction of expression vectors and the expression ofgenes in transfected cells involves the use of molecular cloningtechniques also well known in the art. Sambrook et al., MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., (1989) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., (Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc.) Usefulpromoters for such purposes include a metallothionein promoter, aconstitutive adenovirus major late promoter, a dexamethasone-inducibleMMTV promoter, a SV40 promoter, a MRP polIII promoter, a constitutiveMPSV promoter, a tetracycline-inducible CMV promoter (such as the humanimmediate-early CMV promoter), and a constitutive CMV promoter.

[0122] Methods for transfecting genes into mammalian cells and obtainingtheir expression for in vitro use or for gene therapy, are well known tothe art. See, e.g., Methods in Enzymology, vol. 185, Academic Press,Inc., San Diego, Calif. (D. V. Goeddel, ed.) (1990) or M. Krieger, GeneTransfer and Expression—A Laboratory Manual, Stockton Press, New York,N.Y., (1990).

[0123] Expression vectors useful in this invention depend on theirintended use. Such expression vectors must, of course, containexpression and replication signals compatible with the host cell.Expression vectors useful for expressing the protein of this inventioninclude viral vectors such as alpha viruses, retroviruses, adenovirusesand adeno-associated viruses, plasmid vectors, cosmids, liposomes andthe like. Viral and plasmid vectors are preferred for transfectingmammalian cells. The expression vector pcDNA1 (Invitrogen, San Diego,Calif.), in which the expression control sequence comprises the CMVpromoter, provides good rates of transfection and expression.Adeno-associated viral vectors are useful in the gene therapy methods ofthis invention.

[0124] The construct can also contain a tag to simplify isolation of theprotein. For example, a polyhistidine tag of, e.g., six histidineresidues, can be incorporated at the amino terminal end of the protein.The polyhistidine tag allows convenient isolation of the protein in asingle step by nickel-chelate chromatography.

[0125] In another embodiment, endogenous genes are transcribed byoperatively linking them to expression control sequences suppliedendogenously that recombine with genomic DNA. In one method, oneprovides the cell with a recombinant polynucleotide containing atargeting sequence, which permits homologous recombination into thegenome upstream of the transcriptional start site of target gene; theexpression control sequences; an exon of the target gene; and anunpaired splice-donor site which pairs with a splice acceptor in thetarget gene. Such methods are discussed in Treco et al., WO 94/12650;Treco et al., WO 95/31560 and Treco et al., WO 96/29411.

[0126] The invention also provides recombinant cells comprising anexpression vector for expression of the nucleotide sequences encoding apolypeptide of this invention. Host cells can be selected for highlevels of expression in order to purify the protein. Mammalian cells arepreferred for this purpose, but prokaryotic cells, such as E. coli, alsoare useful. The cell can be, e.g., a recombinant cell in culture or acell in vivo.

[0127] IV. Polynucleotide Probes and Primers

[0128] This invention provides polynucleotide probes and primers thatspecifically hybridize to a sub-sequence of Repro-EN-1.0 cDNA or itscomplement or IB1 cDNA or its complement, under stringent hybridizationconditions. The probes and primers of this invention are polynucleotidesof at least 7 nucleotides, at least 10 nucleotides, at least 15nucleotides, at least 20 nucleotides or at least 25 nucleotides. In oneembodiment, the sequence of the polynucleotide is a contiguous sequencefrom SEQ ID NO:1 or its complement. Any suitable region of theRepro-EN-1.0 or IB1 gene may be chosen as a target for polynucleotidehybridization. Nucleotide substitutions, deletions, and additions may beincorporated into the polynucleotides as long as the characteristicability to specifically hybridize to the target sequence or itscomplement is retained. Nucleotide sequence variation may result fromsequence polymorphisms of various alleles, minor sequencing errors, andthe like.

[0129] The probes and primers of the invention are useful as probes inhybridization assays, such as Southern and northern blots, foridentifying polynucleotides having a nucleotide sequence encoding aRepro-EN-1.0 or IB1 polypeptide, and as primers for amplificationprocedures. The probes and primers of the invention are also useful indetecting the presence, absence or amount of Repro-EN-1.0 or IB1 intissue biopsies and histological sections where the detection method iscarried out in situ, typically after amplification of Repro-EN-1.0 orIB1 sequences using a primer set.

[0130] The probes and primers of this invention also are useful foridentifying allelic forms of Repro-EN-1.0 and animal cognate genes orIB1 and animal cognate genes. Probes and primers can be used to screenhuman or animal genomic DNA or cDNA libraries under, e.g., stringentconditions. DNA molecules that specifically hybridize to the probe arethen further examined to determine whether they are Repro-EN-1.0 allelicvariants or animal cognates or IB1 allelic variants or animal cognates.

[0131] The probes also are useful in oligonucleotide arrays. Such arraysare used in hybridization assays to check the identity of bases in atarget polynucleotide. In essence, when a target hybridizes perfectly toa probe on the array, the target contains the nucleotide sequence of theprobe. When the target hybridizes less well, or does not hybridize atall, then the target and probe differ in sequence by one or morenucleotide. By proper selection of probes, one can check bases on atarget molecule. See, e.g., Chee et al., WO 95/11995. The use theRepro-EN-1.0 or IB1 sequence in genomics is described further below.

[0132] In one embodiment, the polynucleotide is directly or indirectlydetectable through a detectable moiety. A detectable moiety bound toeither an oligonucleotide primer or a probe is subsequently used todetect hybridization of an oligonucleotide primer to the RNA component.Detection of labeled material bound to a Repro-EN-1.0 or IB1polynucleotide in a sample provides a means of determining a diagnosticor prognostic value.

[0133] Although primers and probes can differ in sequence and length,the primary differentiating factor is one of function: primers serve asan initiation point for DNA synthesis of a target polynucleotide, as inRT and PCR reactions, while probes are typically used for hybridizationto and detection of a target polynucleotide. Typical lengths of primersor probes can range from 7-50 nucleotides, preferably from 10-40nucleotides, and most preferably from 15-35 nucleotides. A primer orprobe can also be labeled with a detectable moiety for detection ofhybridization of the primer or probe to the target polynucleotide.

[0134] In general, those of skill in the art recognize that thepolynucleotides used in the invention include both DNA and RNA moleculesand naturally occurring modifications thereof, as well as synthetic,non-naturally occurring analogs of the same, and heteropolymers, ofdeoxyribonucleotides, ribonucleotides, and/or analogs of either. Theparticular composition of a polynucleotide or polynucleotide analog willdepend upon the purpose for which the material will be used and theenvironment in which the material will be placed. Modified or synthetic,non-naturally occurring nucleotides have been designed to serve avariety of purposes and to remain stable in a variety of environments,such as those in which nucleases are present.

[0135] Oligonucleotides preferably are synthesized, e.g., on an AppliedBioSystems or other commercially available oligonucleotide synthesizeraccording to specifications provided by the manufacturer.Oligonucleotides may be prepared using any suitable method, such as thephosphotriester and phosphodiester methods, or automated embodimentsthereof. In one such automated embodiment, diethylphosphoramidates areused as starting materials and may be synthesized as described byBeaucage et al., Tetrahedron Letters 22: 1859 (1981), and U.S. Pat. No.4,458,066.

[0136] Polynucleotides, e.g., probes, also can be recombinantly producedthrough the use of plasmids or other vectors.

[0137] In one aspect this invention provides a probe that specificallyhybridizes to the 5′ untranslated region of Repro-EN-1.0 or IB1, thecoding region of Repro-EN-1.0 or IB1, or a region of Repro-EN-1.0 or IB1encoding an epitope of the Repro-EN-1.0 or IB1 polypeptide.

[0138] In another aspect, this invention provides a primer pair whichamplifies a nucleotide sequence encoding a polypeptide epitope ofRepro-EN-1.0 or IB1 recognized by an antibody from an individualdiagnosed with endometriosis. A primer pair that amplifies a particularnucleotide sequence (given in the 5′ to 3′ orientation) includes a 5′primer and a 3′ primer. The 3′ primer hybridizes to the 3′ end of thenucleotide sequence or downstream from it. The 5′ primer hybridizes tothe 3′ end of the complement of the nucleotide sequence or downstreamfrom it. In this way, the primers can amplify a polynucleotide thatcomprises the nucleotide sequence. One nucleotide sequence encoding apolypeptide epitope of Repro-EN-1.0 has been identified within the about2.2 kb from 3′ end of the coding sequence of Repro-EN-1.0 (SEQ ID NO:1).

[0139] V. Methods for Detecting Repro-EN-1.0 and IB1 Polynucleotides

[0140] The probes and primers of this invention are useful, among otherthings, in detecting Repro-EN-1.0 or IB1 polynucleotides in a sample. Amethod for detecting the presence, absence or amount of a Repro-EN-1.0or IB1 polynucleotide in a sample involves two steps: (1) specificallyhybridizing a polynucleotide probe or primer to a Repro-EN-1.0 or IB1polynucleotide, and (2) detecting the specific hybridization.

[0141] For the first step of the method, the polynucleotide used forspecific hybridization is chosen to hybridize to any suitable region ofRepro-EN-1.0. The polynucleotide can be a DNA or RNA molecule, as wellas a synthetic, non-naturally occurring analog of the same. Thepolynucleotides in this step are polynucleotide primers andpolynucleotide probes disclosed herein. This includes probes and primershaving sequences selected from the sequence of Repro-EN-1.0 (SEQ IDNO:1) or selected from the cyber-sequence of Repro-EN-1.0 (SEQ ID NO:5).

[0142] For the second step of the reaction, any suitable method fordetecting specific hybridization of a polynucleotide to Repro-EN-1.0 orIB1 may be used. Such methods include, e.g., amplification by extensionof a hybridized primer using reverse transcriptase (RT); extension of ahybridized primer using RT-PCR or other methods of amplification; and insitu detection of a hybridized primer. In in situ hybridization, asample of tissue or cells is fixed onto a glass slide and permeablizedsufficiently for use with in situ hybridization techniques. Detectablemoieties used in these methods include, e.g., labeled polynucleotideprobes; direct incorporation of label in amplification or RT reactions,and labeled polynucleotide primers.

[0143] Often, cell extracts or tissue samples used in methods fordetermining the amount of a polynucleotide in a sample will containvariable amounts of cells or extraneous extracellular matrix materials.Thus, a method for determining the cell number in a sample is importantfor determining the relative amount per cell of a test polynucleotidesuch as Repro-EN-1.0 or IB1. A control for cell number and amplificationefficiency is useful for determining diagnostic values for a sample of apotential cancer, and a control is particularly useful for comparing theamount of test polynucleotide such as Repro-EN-1.0 or IB1 in sample to adiagnostic value for breast cancer, uterine cancer or fallopian tubecancer. A preferred embodiment of the control RNA is endogenouslyexpressed 28S rRNA. (See, e.g., Khan et al., Neurosci. Lett. 147:114-117 (1992) which used 28S rRNA as a control, by diluting reversetranscribed 28S rRNA and adding it to the amplification reaction.)

[0144] VI. Inhibitory Polynucleotides for Inhibiting Repro-EN-1.0 andIB1 Expression

[0145] A. General

[0146] This invention also provides inhibitory polynucleotides directedagainst Repro-EN-1.0 or IB1 polynucleotides that inhibit Repro-EN-1.0 orIB1 expression and, therefore inhibit its activity in a cell. Inhibitorypolynucleotides can inhibit Repro-EN-1.0 or IB1 activity in a number ofways. According to one mechanism, the polynucleotide preventstranscription of the Repro-EN-1.0 or IB1 gene (for instance, by triplehelix formation). In another mechanism, the polynucleotide destabilizesthe Repro-EN-1.0 or IB1 and reduces its half-life. In another mechanism,the polynucleotide inhibits assembly of the RNA component into theRepro-EN-1.0 or IB1 by binding to Repro-EN-1.0 or IB1.

[0147] An inhibitory polynucleotide is a polynucleotide that is capableof specifically hybridizing with a target polynucleotide and thatinterferes with the transcription, processing, translation or otheractivity the target polynucleotide. Inhibitory polynucleotides generallyare single-stranded and have a sequence of at least 7, 8, 9, 10, or 11nucleotides capable of specifically hybridizing to the target sequence.RNA sequences generally require a sequence of at least 10 nucleotidesfor specific hybridization. Inhibitory polynucleotides include, withoutlimitation, antisense molecules, ribozymes, sense molecules andtriplex-forming molecules. In one embodiment, the inhibitorypolynucleotide is no more than about 50 nucleotides long.

[0148] While not wishing to be limited by theory, it is believed thatinhibitory polynucleotides inhibit the function of a target, in part, bybinding to the appropriate target sequence. An inhibitory polynucleotidecan inhibit DNA replication or DNA transcription by, for example,interfering with the attachment of DNA or RNA polymerase to the promoterby binding to a transcriptional initiation site or a template. It caninterfere with processing of mRNA, poly(A) addition to mRNA ortranslation of mRNA by, for example, binding to regions of the RNAtranscript such as the ribosome binding site. It can promote inhibitorymechanisms of the cells, such as promoting RNA degradation via RNaseaction. The inhibitory polynucleotide can bind to the major groove ofthe duplex DNA to form a triple helical or “triplex” structure. Methodsof inhibition using inhibitory polynucleotides therefore encompass anumber of different approaches to altering expression of specific genesthat operate by different mechanisms. These different types ofinhibitory polynucleotide technology are described in C. Helene and J.Toulme, (1990) Biochim. Biophys. Acta., 1049:99-125.

[0149] Antisense polynucleotides can include deoxyribonucleotides orribonucleotides. They can be chemically modified so as to improvestability in the body. Properties of the polynucleotide can beengineered to impart stability (e.g., nuclease resistance), tighterbinding or the desired Tm. See, e.g., International patent publicationNo. 94/12633.

[0150] The general approach to constructing various polynucleotidesuseful in inhibitory polynucleotide therapy has been reviewed by A. R.Vander Krol et al. (1988), Biotechniques 6:958-976, and by C. A. Steinet al., (1988) Cancer Res. (1988) 48:2659-2668. See alsoOligodeoxynucleotides: Antisense Inhibitors of Gene Expression, Cohen,J. S., editor, MacMillan Press, London, pages 79-196 (1989), andAntisense RNA and DNA, (1988), D. A. Melton, Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. In certain embodiments inhibitorypolynucleotides comprise a derivatized substituent which issubstantially non-interfering with respect to hybridization of theinhibitory polynucleotide to the target polynucleotide.

[0151] B. Antisense

[0152] This invention provides antisense polynucleotides capable ofspecifically hybridizing to a target sequence of Repro-EN-1.0 or IB1.Antisense polynucleotides are useful in vitro or in vivo to inhibit theactivity of Repro-EN-1.0 or IB1.

[0153] The antisense polynucleotides of this invention comprise anantisense sequence of at least 7 nucleotides that specifically hybridizeto a sequence from Repro-EN-1.0 or IB1 and, more particularly, mammalianRepro-EN-1.0 or IB1 and human Repro-EN-1.0 or IB1.

[0154] The antisense sequence can be between about 10 and about 50nucleotides or between about 15 and about 35 nucleotides. In otherembodiments, antisense polynucleotides are polynucleotides of less thanabout 100 nucleotides or less than about 200 nucleotides. Accordingly, asequence of the antisense polynucleotide can specifically hybridize toall or part of the Repro-EN-1.0 or IB1, such as antisensepolynucleotides to the Repro-EN-1.0 or IB1 gene or its transcribed RNA.In one embodiment, the sequence of the polynucleotide contains within itthe antisense sequence. In this case, the antisense sequence iscontained within a polynucleotide of longer sequence. In anotherembodiment, the sequence of the polynucleotide consists essentially of,or is, the antisense sequence. Thus, for example, the antisensepolynucleotide can be a polynucleotide of less than about 50 nucleotidesin a sequence that specifically hybridizes to the target sequence.

[0155] Generally, to assure specific hybridization, the antisensesequence is substantially complementary to the target sequence inRepro-EN-1.0 or IB1. In certain embodiments, the antisense sequence isexactly complementary to the target sequence. The antisensepolynucleotides may include nucleotide substitutions, additions,deletions, or transpositions, so long as specific binding to therelevant target sequence corresponding to Repro-EN-1.0 mRNA or its gene,or IB1 mRNA or its gene, is retained as a functional property of thepolynucleotide.

[0156] The antisense polynucleotide should be long enough to form astable duplex but short enough, depending on the mode of delivery, toadminister in vivo, if desired. The minimum length of a polynucleotiderequired for specific hybridization to a target sequence depends onseveral factors, such as G/C content, positioning of mismatched bases(if any), degree of uniqueness of the sequence as compared to thepopulation of target polynucleotides, and chemical nature of thepolynucleotide (e.g., methylphosphonate backbone, polyamide nucleicacid, phosphorothioate, etc.), among others.

[0157] For general methods relating to antisense polynucleotides, seeAntisense RNA and DNA, (1988), D. A. Melton, Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). For a review of antisensetherapy, see, e.g., Uhlmann et al., Chem. Reviews, 90:543-584 (1990).

[0158] C. Ribozymes

[0159] Cleavage of Repro-EN-1.0 or IB1 can be induced by the use ofribozymes or catalytic RNA. In this approach, the ribozyme would containeither naturally occurring RNA (ribozymes) or synthetic nucleic acidswith catalytic activity. Bratty et al., (1992) Biochim. Biophys. Acta.,1216:345-59 (1993) and Denhardt, (1992) Ann. N.Y. Acad. Sci., 660:70-76describe methods for making ribozymes.

[0160] Unlike the antisense and other polynucleotides described above,which bind to an RNA or a DNA, a ribozyme not only binds but alsospecifically cleaves and thereby potentially inactivates a target RNA.Such a ribozyme can comprise 5′- and 3′-terminal sequences complementaryto the Repro-EN-1.0 RNA or IB1 RNA.

[0161] Optimum target sites for ribozyme-mediated inhibition of activitycan be determined as described by Sullivan et al., PCT patentpublication No. 94/02595 and Draper et al., PCT patent publication No.93/23569. As described by Hu et al., PCT patent publication No.94/03596, antisense and ribozyme functions can be combined in a singlepolynucleotide. Upon review of the RNA sequence of Repro-EN-1.0 and IB1,those in the art will note that several useful ribozyme target sites arepresent and susceptible to cleavage by, for example, a hammerhead motifribozyme.

[0162] Such engineered ribozymes can be expressed in cells or can betransferred by a variety of means (e.g., liposomes, immunoliposomes,biolistics, direct uptake into cells, etc.). Other forms of ribozymes(group I intron ribozymes (Cech (1995) Biotechnology 13; 323);hammerhead ribozymes (Edgington (1992) Biotechnology 10: 256) can beengineered on the basis of the disclosed Repro-EN-1.0 or IB1 sequenceinformation to catalyze cleavage of Repro-EN-1.0 RNA or IB1 RNA.Moreover, ribozymes can comprise one or more modified nucleotides ormodified linkages between nucleotides, as described above.

[0163] D. Other Inhibitory Polynucleotides

[0164] In addition to the antisense and ribozyme inhibitorypolynucleotides, one can construct polynucleotides that will bind toduplex nucleic acid either in the folded RNA component or in the genefor the RNA component, forming a triple helix-containing or triplexnucleic acid to inhibit Repro-EN-1.0 or IB1 activity. Suchpolynucleotides of the invention are constructed using the base-pairingrules of triple helix formation and the nucleotide sequence of the RNAcomponent (Cheng et al. (1988) J. Biol. Chem. 263: 15110; Ferrin andCamerini-Otero (1991) Science 354: 1494; Ramdas et al. (1989) J. Biol.Chem. 264: 17395; Strobel et al. (1991) Science 254: 1639; Hsieh et al.(1990) op.cit.; Rigas et al. (1986) Proc. Natl. Acad. Sci. (U.S.A.) 83:9591. Such polynucleotides can block Repro-EN-1.0 or IB1 activity in anumber of ways, including by preventing transcription of theRepro-EN-1.0 or IB1 gene.

[0165] Typically, and depending on mode of action, the triplex-formingpolynucleotides of the invention comprise a sequence large enough toform a stable triple helix but small enough, depending on the mode ofdelivery, to administer in vivo.

[0166] E. Methods for Making Inhibitory Polynucleotides

[0167] Inhibitory polynucleotides can be made chemically orrecombinantly.

[0168] 1. Chemical Synthesis

[0169] Small inhibitory polynucleotides for direct delivery can be madeby chemical synthesis. Chemically synthesized polynucleotides can be DNAor RNA, or can include nucleotide analogs or backbones that are notlimited to phosphodiester linkages.

[0170] 2. Recombinant Production

[0171] For delivery into cells or for gene therapy methods, recombinantproduction of inhibitory polynucleotides through the use of expressionvectors is particularly useful. Accordingly, this invention alsoprovides expression vectors, e.g., recombinant polynucleotide moleculescomprising expression control sequences operatively linked to thenucleotide sequence encoding the inhibitory polynucleotide.

[0172] VII. Repro-EN-1.0 and IB1 Polypeptides

[0173] This invention also provides purified, recombinant Repro-EN-1.0and IB1 polypeptide, and Repro-EN-1.0 and IB1 analogs. RecombinantRepro-EN-1.0 polypeptide includes the polypeptide whose amino acidsequence is presented in SEQ ID NO:2, as well as allelic variants of it.Repro-EN-1.0 analogs include 1) immunogenic fragments of Repro-EN-1.0;2) homologs of Repro-EN-1.0 from other mammals (especially primates); 3)fragments of Repro-EN-1.0 comprising at least 5 consecutive amino acidsfrom the sequence of Repro-EN-1.0; 4) non-naturally occurringpolypeptides whose sequences are substantially identical toRepro-EN-1.0; and 5) fusion proteins comprising a Repro-EN-1.0 or aRepro-EN-1.0 analog fused to a second polypeptide moiety.

[0174] Repro-EN-1.0 polypeptide refers to native Repro-EN-1.0, thepolypeptide whose amino acid sequence is the amino acid sequence of SEQID NO:2, and to allelic variants of it. Polynucleotide molecules thatencode allelic variants of Repro-EN-1.0 are isolatable from endometrialcancer cell cDNA or genomic DNA and typically hybridize under stringentconditions to the nucleotide sequence encoding Repro-EN-1.0 (SEQ IDNO:1). They can be obtained by amplification using, e.g., PCR primerstaken from the sequence of Repro-EN-1.0 described herein.

[0175] Repro-EN-1.0 polypeptides are useful as immunogens to elicit theproduction of anti-Repro-EN-1.0 antibodies, as affinity capturemolecules to isolate such antibodies from a mixture, and as controls indiagnostic methods aimed at detecting Repro-EN-1.0 in a sample.

[0176] Immunogenic Repro-EN-1.0 analogs are polypeptides having asequence of at least 5 amino acids selected from native Repro-EN-1.0 andwhich, when presented to an animal as an immunogen, elicit a humoral orcell-mediated immune response. This includes polypeptides comprising anamino acid sequence which is an epitope from Repro-EN-1.0, such asimmunogenic fragments of Repro-EN-1.0. Repro-EN-1.0 protein analogsoptionally are in isolated form. Persons skilled in the art are familiarwith methods of identifying probable epitopes of a protein. For example,polypeptide fragments most likely to elicit an immune response againstthe native protein are those that exist on the surface of the nativeprotein. Portions of a protein on the surface tend to includehydrophilic amino acids. Such regions can be identified by inspection orwith available software. In one embodiment immunogenic polypeptide is apolypeptide comprising a sequence of at least 5 consecutive hydrophilicamino acids, or at least five hydrophilic amino acids in a series ofeight consecutive amino acids. Repro-EN-1.0 contains long hydrophilicstretches. Examples of such polypeptides include those comprising thesequences: KTPSAEERR (SEQ ID NO:7), RARPESER (SEQ ID NO:8), RMSDMLSR(SEQ ID NO:9) or NEKLSPKPG (SEQ ID NO:10).

[0177] The cDNA encoding Repro-EN-1.0 of SEQ ID NO:1 was discovered byscreening an expression library of cDNA from an endometrial carcinomacell line with serum pooled from subjects diagnosed with endometriosis.Therefore, polypeptides comprising an epitope of Repro-EN-1.0 can beidentified by screening with such serum. Preferably, the test serum is aserum pooled from several subjects positively diagnosed withendometriosis. At least one epitope of Repro-EN-1.0 exists in a fragmentof 567 amino acids from the carboxy-terminus of the molecule (aminoacids 293-860 of SEQ ID NO:2). At least one epitope of IB1 exists in afragment of 644 amino acids from the carboxy terminus of the molecule(amino acids 293-937 of SEQ ID NO: 4). Immunogenic fragments are useful,for example, to detect the presence of antibodies against Repro-EN-1.0or IB1 in patient serum samples. This test is useful in diagnosisbecause the presence of such antibodies is a diagnostic marker forendometriosis.

[0178] Fragments of Repro-EN-1.0 or IB1 include those having at least 5amino acids, at least 10 amino acids, at least 50 amino acids, at least100 amino acids or at least 200 amino acids in a sequence fromRepro-EN-1.0 or IB1. Fragments are useful as immunogens to produce animmune response against Repro-EN-1.0 or IB1 in the production ofantibodies. Alternatively, fragments having appropriate amino acidmotifs are useful as agretopes to bind with MHC molecules. Suchcomplexes are useful in inducing anergy against Repro-EN-1.0 or IB1.

[0179] Non-naturally occurring analogs of Repro-EN-1.0 or IB1 have atleast 90% sequence identity with Repro-EN-1.0 or IB1. They can be madeby, for example, introducing conservative amino acid substitutions intothe sequence of Repro-EN-1.0 or IB1. Such molecules are useful as decoysor as active analogs.

[0180] Homologs of Repro-EN-1.0 or IB1 from other animals generally havesequences that are substantially identical to that of SEQ ID NO:1 or SEQID NO:3. Genomic DNA or cDNA encoding them can be identified throughscreening libraries under stringent hybridization conditions using aRepro-EN-1.0 or IB1 probe of this invention.

[0181] Fusion proteins include a fragment of Repro-EN-1.0 or IB1 fusedto a second polypeptide moiety at the carboxy or amino terminus. Thesecond polypeptide can function, for example, as a detectable label.Such markers include fluorescent protein, enzyme marker of protein froma two-hybrid system.

[0182] Repro-EN-1.0 or IB1 and analogs are most easily producedrecombinantly, as described herein. Recombinant Repro-EN-1.0 or IB1 canbe purified by affinity purification. In one method, recombinantRepro-EN-1.0 or IB1 analogs comprise a polyhistidine tag. The protein ispurified on a nickel-chelate affinity matrix. In another method,Repro-EN-1.0 or IB1 is purified using an affinity matrix carryinganti-Repro-EN-1.0 or IB1 antibodies.

[0183] VIII. Antibodies and Hybridomas

[0184] In one aspect this invention provides a composition comprising anantibody that specifically binds Repro-EN-1.0 or IB1 polypeptides.Antibodies preferably have affinity of at least 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸M⁻¹, or 10⁹ M⁻¹. This invention contemplates both polyclonal andmonoclonal antibody compositions.

[0185] In one embodiment this invention provides immunotoxins againstRepro-EN-1.0- or IB1-expressing cells. Immunotoxins are antibodies andthe like as described herein coupled to a compound, e.g., a toxin, thatis toxic to a target cell. Toxins can include, for example, radioactiveisotopes, ricin, cisplatin, antisense molecules, Diphtheria toxin,Pseudomonas exotoxin A or Bacillus anthraces protective antigen.Immunotoxins bind to cancer cells that express Repro-EN-1.0 or IB1 andkill them. They are useful in the therapeutic methods of this invention.The antibodies of the invention have many uses. For example, suchantibodies are useful for detecting Repro-EN-1.0 or IB1 polypeptides inimmunoassays. The antibodies also can be used to screen expressionlibraries for particular expression products such as mammalianRepro-EN-1.0 or IB1. These are useful for detecting or diagnosingvarious pathological conditions related to the presence of therespective antigens. Usually the antibodies in such a procedure arelabeled with a moiety allowing easy detection of presence of antigen byantibody binding. Antibodies raised against Repro-EN-1.0 or IB1 can alsobe used to raise anti-idiotypic antibodies.

[0186] A. Production of Antibodies

[0187] A number of immunogens are used to produce antibodies thatspecifically bind Repro-EN-1.0 or IB1 polypeptides. Full-lengthRepro-EN-1.0 or IB1 is a suitable immunogen. Typically, the immunogen ofinterest is a peptide of at least about 3 amino acids, more typicallythe peptide is 5 amino acids in length, preferably, the fragment is 10amino acids in length and more preferably the fragment is 15 amino acidsin length or greater. The peptides can be coupled to a carrier protein(e.g., as a fusion protein), or are recombinantly expressed in animmunization vector. Antigenic determinants on peptides to whichantibodies bind are typically 3 to 10 amino acids in length. Naturallyoccurring polypeptides are also used either in pure or impure form.

[0188] Recombinant polypeptides are expressed in eukaryotic orprokaryotic cells and purified using standard techniques. Thepolypeptide, or a synthetic version thereof, is then injected into ananimal capable of producing antibodies. Either monoclonal or polyclonalantibodies can be generated for subsequent use in immunoassays tomeasure the presence and quantity of the polypeptide.

[0189] Methods for producing polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purifiedpolypeptide, a polypeptide coupled to an appropriate carrier (e.g., GST,keyhole limpet hemanocyanin, etc.), or a polypeptide incorporated intoan immunization vector such as a recombinant vaccinia virus (see, U.S.Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunizedwith the mixture. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the polypeptide of interest. When appropriately hightiters of antibody to the immunogen are obtained, blood is collectedfrom the animal and antisera are prepared. Further fractionation of theantisera to enrich for antibodies reactive to the polypeptide isperformed where desired. See, e.g., Coligan (1991) Current Protocols inImmunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: ALaboratory Manual Cold Spring Harbor Press, NY.

[0190] Antibodies, including binding fragments and single chainrecombinant versions thereof, against predetermined fragments ofRepro-EN-1.0 or IB1 proteins are raised by immunizing animals, e.g.,with conjugates of the fragments with carrier proteins as describedabove.

[0191] Monoclonal antibodies are prepared from cells secreting thedesired antibody. These antibodies are screened for binding to normal ormodified polypeptides, or screened for agonistic or antagonisticactivity, e.g., activity mediated through Repro-EN-1.0 or IB1. In someinstances, it is desirable to prepare monoclonal antibodies from variousmammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4thed.) Lange Medical Publications, Los Altos, Calif., and references citedtherein; Harlow and Lane, Supra; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256: 495-497.

[0192] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors. See, Huse et al.(1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341:544-546.

[0193] Also, recombinant immunoglobulins may be produced. See, Cabilly,U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci.USA 86: 10029-10033.

[0194] Frequently, the polypeptides and antibodies will be labeled byjoining, either covalently or non-covalently, a substance which providesfor a detectable signal. A wide variety of labels and conjugationtechniques are known and are reported extensively in both the scientificand patent literature. Thus, an antibody used for detecting an analytecan be directly labeled with a detectable moiety, or may be indirectlylabeled by, for example, binding to the antibody a secondary antibodythat is, itself directly or indirectly labeled.

[0195] The antibodies of this invention are also used for affinitychromatography in isolating Repro-EN-1.0 or IB1 proteins. Columns areprepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified Repro-EN-1.0 orIB1 polypeptides are released.

[0196] An alternative approach is the generation of humanizedimmunoglobulins by linking the CDR regions of non-human antibodies tohuman constant regions by recombinant DNA techniques. See Queen et al.,U.S. Pat. No. 5,585,089.

[0197] A further approach for isolating DNA sequences which encode ahuman monoclonal antibody or a binding fragment thereof is by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989) and then cloningand amplifying the sequences which encode the antibody (or bindingfragment) of the desired specificity. The protocol described by Huse isrendered more efficient in combination with phage display technology.See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047.Phage display technology can also be used to mutagenize CDR regions ofantibodies previously shown to have affinity for Repro-EN-1.0 or IB1protein receptors or their ligands. Antibodies having improved bindingaffinity are selected.

[0198] In another embodiment of the invention, fragments of antibodiesagainst Repro-EN-1.0 or IB1 protein or protein analogs are provided.Typically, these fragments exhibit specific binding to the Repro-EN-1.0protein receptor similar to that of a complete immunoglobulin. Antibodyfragments include separate heavy chains, light chains Fab, Fab′ F(ab′)₂and Fv. Fragments are produced by recombinant DNA techniques, or byenzymic or chemical separation of intact immunoglobulins.

[0199] IX. Methods for Detecting Repro-EN-1.0 and IB1 Polypeptides

[0200] Repro-EN-1.0 or IB1 polypeptides can be identified by any methodsknown in the art. In one embodiment, the methods involve detecting thepolypeptide with a ligand that specifically recognizes the polypeptide(e.g., an immunoassay). The antibodies of the invention are particularlyuseful for specific detection of Repro-EN-1.0 or IB1 polypeptides. Avariety of antibody-based detection methods are known in the art. Theseinclude, for example, radioimmunoassay, sandwich immunoassays (includingELISA), immunofluorescent assays, western blot, affinity chromatography(affinity ligand bound to a solid phase), and in situ detection withlabeled antibodies. Another method for detecting Repro-EN-1.0 or IB1polypeptides involves identifying the polypeptide according to its massthrough, for example, gel electrophoresis, mass spectrometry or HPLC.Subject samples can be taken from any number of appropriate sources,such as saliva, peritoneal fluid, blood or a blood product (e.g.,serum), urine, tissue biopsy (e.g., lymph node tissue), etc.

[0201] a. Immunoassays

[0202] The present invention also provides methods for detection ofRepro-EN-1.0 or IB1 polypeptides employing one or more anti-Repro-EN-1.0or IB1 antibody reagents (i.e., immunoassays). A number of wellestablished immunological binding assay formats suitable for thepractice of the invention are known (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, e.g., METHODS INCELL BIOLOGY VOLUME 37: ANTIBODIES IN CELL BIOLOGY, Asai, ed. AcademicPress, Inc. New York (1993); BASIC AND CLINICAL IMMUNOLOGY 7th Edition,Stites & Terr, eds. (1991); Harlow and Lane, supra [e.g., Chapter 14],and Ausubel et al., supra, [e.g., Chapter 11]. Typically, immunologicalbinding assays (or immunoassays) utilize a “capture agent” tospecifically bind to and, often, immobilize the analyte to a solidphase. In one embodiment, the capture agent is a moiety thatspecifically binds to a Repro-EN-1.0 or IB1 polypeptide or subsequence,such as an anti-Repro-EN-1.0 or anti-IB1 antibody.

[0203] Usually the Repro-EN-1.0 or IB1 polypeptide being assayed isdetected directly or indirectly using a detectable label. The particularlabel or detectable group used in the assay is usually not a criticalaspect of the invention, so long as it does not significantly interferewith the specific binding of the antibody or antibodies used in theassay. The label may be covalently attached to the capture agent (e.g.,an anti-Repro-EN-1.0 or or anti-IB1 antibody), or may be attached to athird moiety, such as another antibody, that specifically binds to theRepro-EN-1.0 polypeptide.

[0204] The present invention provides methods and reagents forcompetitive and noncompetitive immunoassays for detecting Repro-EN-1.0or IB1 polypeptides. Non-competitive immunoassays are assays in whichthe amount of captured analyte (in this case Repro-EN-1.0 or IB1) isdirectly measured. One such assay is a two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on the Repro-EN-1.0 or IB1 protein. See, e.g.,Maddox et al., 1983, J. Exp. Med., 158:1211 for background information.In one preferred “sandwich” assay, the capture agent (e.g., ananti-Repro-EN-1.0 or anti-IB1 antibody) is bound directly to a solidsubstrate where it is immobilized. These immobilized antibodies thencapture any Repro-EN-1.0 or IB1 protein present in the test sample. TheRepro-EN-1.0 or IB1 polypeptide thus immobilized can then be labeled,i.e., by binding to a second anti-Repro-EN-1.0 or IB1 antibody bearing alabel. Alternatively, the second anti-Repro-EN-1.0 or IB1 antibody maylack a label, but be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond antibody alternatively can be modified with a detectable moiety,such as biotin, to which a third labeled molecule can specifically bind,such as enzyme-labeled streptavidin.

[0205] In competitive assays, the amount of Repro-EN-1.0 or IB1 proteinpresent in the sample is measured indirectly by measuring the amount ofan added (exogenous) Repro-EN-1.0 or IB1 displaced (or competed away)from a capture agent (e.g., anti-Repro-EN-1.0 or anti-IB1 antibody) bythe Repro-EN-1.0 or IB1 protein present in the sample.

[0206] A hapten inhibition assay is another example of a competitiveassay. In this assay Repro-EN-1.0 or IB1 protein is immobilized on asolid substrate. A known amount of anti-Repro-EN-1.0 or anti-IB1antibody is added to the sample, and the sample is then contacted withthe immobilized Repro-EN-1.0 or IB1 protein. In this case, the amount ofanti-Repro-EN-1.0 or anti-IB1 antibody bound to the immobilizedRepro-EN-1.0 or IB1 protein is inversely proportional to the amount ofRepro-EN-1.0 protein present in the sample. The amount of immobilizedantibody may be detected by detecting either the immobilized fraction ofantibody or the fraction of the antibody that remains in solution. Inthis aspect, detection may be direct, where the antibody is labeled, orindirect where the label is bound to a molecule that specifically bindsto the antibody as described above.

[0207] b. Other Antibody-Based Assay Formats

[0208] The invention also provides reagents and methods for detectingand quantifying the presence of Repro-EN-1.0 or IB1 polypeptide in thesample by using an immunoblot (Western blot) format. Another immunoassayis the so-called “lateral flow chromatography.” In a non-competitiveversion of lateral flow chromatography, a sample moves across asubstrate by, e.g., capillary action, and encounters a mobile labeledantibody that binds the analyte forming a conjugate. The conjugate thenmoves across the substrate and encounters an immobilized second antibodythat binds the analyte. Thus, immobilized analyte is detected bydetecting the labeled antibody. In a competitive version of lateral flowchromatography a labeled version of the analyte moves across the carrierand competes with unlabeled analyte for binding with the immobilizedantibody. The greater the amount of the analyte in the sample, the lessthe binding by labeled analyte and, therefore, the weaker the signal.See, e.g., May et al., U.S. Pat. Nos. 5,622,871 and Rosenstein,5,591,645.

[0209] c. Solid Phases: Substrates, Solid Supports, Membranes, Filters

[0210] As noted supra, depending upon the assay, various components,including the antigen, target antibody, or anti-Repro-EN-1.0 or anti-IB1antibody, may be bound to a solid surface or support (i.e., a substrate,membrane, or filter paper). Many methods for immobilizing biomoleculesto a variety of solid surfaces are known in the art. For instance, thesolid surface may be a membrane (e.g., nitrocellulose), a microtiterdish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass orplastic), a dipstick (e.g. glass, PVC, polypropylene, polystyrene,latex, and the like), a microcentrifuge tube, or a glass or plasticbead. The desired component may be covalently bound or noncovalentlyattached through nonspecific bonding.

[0211] A wide variety of organic and inorganic polymers, both naturaland synthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

[0212] d. Mass Spectrometry

[0213] The mass of a molecule frequently can be used as an identifier ofthe molecule. Therefore, methods of mass spectrometry can be used toidentify a protein analyte. Mass spectrometers can measure mass bydetermining the time required for an ionized analyte to travel down aflight tube and to be detected by an ion detector.

[0214] One method of mass spectrometry for proteins is matrix-assistedlaser desorption/ionization mass spectrometry (“MALDI”). In MALDI theanalyte is mixed with an energy absorbing matrix material that absorbsenergy of the wavelength of a laser and placed on the surface of aprobe. Upon striking the matrix with the laser, the analyte is desorbedfrom the probe surface, ionized, and detected by the ion detector. See,for example, Hillenkamp et al., U.S. Pat. No. 5,118,937.

[0215] Other methods of mass spectrometry for proteins are described inHutchens and Yip, U.S. Pat. No. 5,719,060. In one such method referredto as Surfaces Enhanced for Affinity Capture (“SEAC”) a solid phaseaffinity reagent that binds the analyte specifically ornon-specifically, such as an antibody or a metal ion, is used toseparate the analyte from other materials in a sample. Then the capturedanalyte is desorbed from the solid phase by, e.g., laser energy,ionized, and detected by the detector.

[0216] e. Assay Combinations

[0217] The diagnostic and prognostic assays described herein can becarried out in various combinations and can also be carried out inconjunction with other diagnostic or prognostic tests. For example, whenthe present methods are used to diagnose endometriosis, the presence ofa Repro-EN-1.0 or IB1 polypeptide can be used to determine the stage ofthe disease. Tests that may provide additional information includemicroscopic analysis of biopsy samples, detection of antigens (e.g.,cell-surface markers) associated with endometriosis (e.g., usinghistocytochemistry, FACS, or the like).

[0218] X. Diagnostic, Monitoring and Prognostic Methods

[0219] A. Methods of Diagnosing Endometriosis

[0220] We have detected circulating antibodies against Repro-EN-1.0 orIB1 in the blood of women diagnosed with endometriosis. This supportsthe idea that endometriosis has an autoimmune component. Further,Repro-EN-1.0 or IB1 and auto-antibodies against Repro-EN-1.0 or IB1represent two targets in the diagnosis of endometriosis.

[0221] Repro-EN-1.0 or IB1 that is shed into the peritoneal fluid ofwomen with endometriosis is useful in methods of diagnosingendometriosis. These methods include detecting Repro-EN-1.0 or IB1 in abiological sample of a subject. Suitable samples include, withoutlimitation, saliva, blood or a blood product (e.g., serum), urine,menstrual fluid, vaginal secretion and, in particular, peritoneal fluid.Repro-EN-1.0 or IB1 can be detected by any of the methods describedherein. Any detection of Repro-EN-1.0 or IB1 above a normal range is apositive sign in the diagnosis of endometriosis.

[0222] In another aspect, this invention provides methods for diagnosingendometriosis in a subject by detecting in a sample from the subject adiagnostic amount of an antibody that specifically binds to Repro-EN-1.0or IB1 polypeptide. Suitable patient samples include, withoutlimitation, saliva, blood or a blood product (e.g., serum), peritonealfluid, urine, menstrual fluid, vaginal secretion. The antibodies can bedetected by any of the methods for detecting proteins described herein.However, sandwich type assays are particularly useful. In one version,all antibodies are captured onto a solid phase, for example usingprotein A, and antibodies specific for Repro-EN-1.0 or IB1 are detectedusing a directly or indirectly labeled Repro-EN-1.0 or IB1 orpolypeptide fragment of it having an epitope of Repro-EN-1.0 or IB1. Inanother version of the assay, Repro-EN-1.0 or IB1 or an antigenicfragment of it can be used as the capture molecule and capturedantibodies can be detected.

[0223] While the detection of antibodies, in general, againstRepro-EN-1.0 or IB1 is a positive sign of endometriosis, IgE an IgG₄class antibodies are particularly specific and sensitive for thediagnosis of endometriosis. Therefore, in one embodiment, the diagnosticmethod involves specifically detecting IgE or IgG₄ antibodies thatspecifically recognize Repro-EN-1.0 or IB1. Anti-human IgE antibodiesand anti-human IgG₄ antibodies can be easily bought or made.

[0224] 1. In vivo Diagnosis

[0225] In another method of the invention, endometriosis can bediagnosed in vivo. The methods involve detecting Repro-EN-1.0 or IB1 inthe body, e.g., in the peritoneum. In general, any conventional methodfor visualizing diagnostic imaging can be used.

[0226] In one method for diagnosing endometriosis, detection isperformed by laparoscopy. A ligand specific for Repro-EN-1.0 or IB1 isintroduced into the subject at the site of a suspected lesion andbinding is detected using the laparoscope. Alternatively, the bindingcan be detected by, for example, magnetic resonance imaging (MRI) orelectron spin resonance (ESR). Usually gamma-emitting andpositron-emitting radioisotopes are used for camera imaging andparamagnetic isotopes are used for magnetic resonance imaging. Anyamount of binding above background is a positive sign of endometriosis.Persons of skill in the art recognize that not every positive signresults in a definitive diagnosis of a disease.

[0227] Endometriotic lesions can be removed surgically. However, lesionsmay be tiny, and difficult to identify by eye. This invention takesadvantage of Repro-EN-1.0 or IB1 as marker for endometriosis byproviding a method to identify and remove endometriotic lesions. Themethod involves identifying endometriotic lesions in situ using alabeled probe directed to Repro-EN-1.0 or IB1. Then, the lesions areremoved surgically.

[0228] In the practice of this method, a probe is provided. The probebinds to Repro-EN-1.0 or IB1 and is labeled with a detectable markerthat can be detected in a surgical procedure. In particular, the probecan be an antibody that specifically binds Repro-EN-1.0 or IB1.Preferred labels that can be detected during surgery are radioactivelabels and fluorescent labels. Radioactive labels can be detected withthe use of, e.g., a Geiger counter. Fluorescent labels, such as FITC,can be detected using, e.g., a D-Light-System (Storz, TuttlingenGermany).

[0229] Surgery can proceed as follows. The labeled probe is introducedinto the peritoneum of the patient for a time sufficient for the labelto bind to endometriotic lesions. Unbound labeled probe is washed out.Then, endometriotic lesions are identified using a suitable detector.For example, in laparoscopic surgery, a Geiger counter may be introducedthrough the incision. Radioactive (“hot”) spots indicate bound labeledand, therefore, an endometriotic lesion. These lesions are then removedfrom the patient.

[0230] B. Methods of Diagnosing Cancer

[0231] Repro-EN-1.0 or IB1 is up-regulated in breast cancer cells,uterine cancer cells, and prostate cancer cells. Therefore, Repro-EN-1.0or IB1 is a marker for these pathologic conditions. Accordingly, themethods described herein for detecting Repro-EN-1.0 or IB1polynucleotides or Repro-EN-1.0 or IB1 polypeptides in a sample areuseful in methods for diagnosing these cancers, monitoring theirprogress or treatment, and determining patient prognosis. The methods ofthe present invention allow cancerous conditions to be detected withincreased confidence and at an earlier stage, before cells are detectedas cancerous based on pathological characteristics. It is, of course,understood by diagnosticians that diagnostic tests are measured by theirdegree of specificity and sensitivity. Tests which are not perfectlyspecific or sensitive are, nevertheless, useful in diagnosis becausethey provide useful information which, in combination with otherevidence, can provide a definitive diagnosis or indicate a course oftreatment.

[0232] Methods for diagnosis involve determining a diagnostic amount ofRepro-EN-1.0 or IB1 (e.g., mRNA, cDNA or polypeptide) in a patientsample and comparing that amount with a normal range (e.g., a controlamount) expected to be found in the sample. The samples used todetermine the normal range of Repro-EN-1.0 or IB1 can be normal samplesfrom the individual to be tested, or normal samples from otherindividuals not suffering from the disease condition.

[0233] A variety of patient samples can be used in the methods of theinvention. For example, cell extracts, cultured cells, or tissue samplesprovide convenient samples for use with the methods of the invention.The methods of the invention can use samples either in solution orextracts, for example, with RT-PCR, or samples such as tissue sectionsfor in situ methods of detection. Samples can also be obtained fromsources such as fine-needle biopsies, e.g., from breast, uterus orprostate; cellular materials; whole cells; tissue and cell extracts; RNAextracted from tissue and cells and histological sections of tissue.

[0234] Methods for monitoring the course of a cancer with whichRepro-EN-1.0 or IB1 is associated involve determining the amount ofRepro-EN-1.0 or IB1 in a sample at a first and second time. The timescan be during routine physical examinations or during a course oftreatment for the cancer. As cancer appears and/or progresses, theamount of Repro-EN-1.0 or IB1 in a sample is expected to increase.Regression or cure of the cancer are accompanied by a decrease orelimination of Repro-EN-1.0 or IB1 in a sample.

[0235] The diagnostic and prognostic methods can also be carried out inconjunction with other diagnostic or prognostic tests. In someinstances, such combination tests can provide useful informationregarding the progression of a disease, although the present methods fortesting for Repro-EN-1.0 or IB1 provide much useful information in thisregard.

[0236] Another diagnostic method of the invention involves theadministration to a subject of a labeled composition that specificallybinds to cells bearing Repro-EN-1.0, or IB1 such as labelled antibodies.Then, the localization of the label is determined by any of the knownradiologic methods. Any conventional method for visualizing diagnosticimaging can be used. Usually gamma- and positron-emitting radioisotopesare used for camera imaging and paramagnetic isotopes are used for MRI.

[0237] C. Methods of Diagnosing Chromosomal Changes

[0238] The Repro-EN-1.0 or IB1 gene is located on chromosome 1. Atranslocation at this site can result in alteration of Repro-EN-1.0 orIB1 activity, such as activated transcription or changed function.Chromosomal translocations in the vicinity of the Repro-EN-1.0 or IB1gene can be detected by hybridizing a labeled probe of this invention toa chromosome spread. A translocation, duplication or deletion can beidentified by aberrant hybridization patterns compared to normal. Suchtests are useful in detecting genetic abnormalities such as familialdisposition to breast, uterine or prostate cancer, or early onset of thedisease. A method for fluorescent in situ hybridization of chromosomesis provided in the Examples.

[0239] The present invention also provides for kits for performing thediagnostic and prognostic method of the invention. Such kits include apolynucleotide probe or primer, or an antibody specific for Repro-EN-1.0or IB1 and instructions to use the reagents to detect Repro-EN-1.0 orIB1 in a patient sample.

[0240] XI. Methods for Inhibiting Repro-EN-1.0 or IB1 Expression orActivity and of Treating Cancer

[0241] Inhibiting Repro-EN-1.0 or IB1 expression or activity in abreast, uterine or prostate cancer cell can alter the rate of growth oraggressiveness of the cancer. Inhibiting Repro-EN-1.0 or IB1 expressionor activity is useful in vivo in the prophylactic and therapeutictreatment of prostate cancer or other conditions involving Repro-EN-1.0or IB1 expression. Accordingly, this invention provides methods forinhibiting Repro-EN-1.0 or IB1 expression or activity. The methodsinvolve contacting a prostate cancer cell, in vitro or in vivo, with aninhibitory polynucleotide, an immunotoxin or another compound thatinhibits Repro-EN-1.0 or IB1 expression or activity.

[0242] A. Delivery of Inhibitory Polynucleotides

[0243] This invention contemplates a variety of means for delivering aninhibitory polynucleotide to a subject including, for example, directuptake of the molecule by a cell from solution, facilitated uptakethrough lipofection (e.g., liposomes or immunoliposomes),particle-mediated transfection, and intracellular expression from anexpression cassette having an expression control sequence operablylinked to a nucleotide sequence that encodes the inhibitorypolynucleotide. Methods useful for delivery of polynucleotides fortherapeutic for therapeutic purposes are described in Inouye et al, U.S.Pat. No. 5,272,065.

[0244] B. Pharmaceutical Compositions and Treatment

[0245] Agents, such as inhibitory polynucleotides, immunotoxins or othercompounds that inhibit Repro-EN-1.0 or IB1 expression or activitypreferably are delivered in pharmaceutical compositions comprising theagent and a pharmaceutically acceptable carrier. The agent can beadministered by any route that gives it access to cells expressingRepro-EN-1.0 or IB1, for example, prostate tumor cells. This includes,for example, aqueous solutions for enteral, parenteral or transmucosaladministration, e.g., for intravenous administration, as tonics andadministration to mucous or other membranes as, for example, nose or eyedrops; solid and other non-aqueous compositions for enteral ortransdermal delivery, e.g., as pills, tablets, powders or capsules;transdermal or transmucosal delivery systems for topical administration,and aerosols or mists for delivery by inhalation. One advantage ofdelivery by a mode that is easy to administer, e.g., enteral or byintravenous or intramuscular injection is that such modes mimic possiblemodes of delivery should the agent be formulated as a pharmaceutical.

[0246] In one embodiment, the pharmaceutical composition is in the formof a unit dose which contains a pharmacologically effective amount ofthe Repro-EN-1.0 or IB1 inhibitory compound. The unit dose, taken aspart of a therapeutic regimen, results in inhibition of growth ofprostate cancer cells. Thus, the pharmaceutical compositions of theinvention, whatever the form, are administered in a pharmacologicallyeffective amount to the subject.

[0247] The amount of the pharmaceutical composition delivered, the modeof administration and the time course of treatment are at the discretionof the treating physician. Prophylactic treatments are indicated forpersons at higher than average risk of getting prostate cancer, breastcancer or uterine cancer. For example, persons with elevated PSA, PAP(prostate acid phosphatase) or PSP (prostate specific protein) levelsare at increased risk of prostate cancer. Persons who have the BRCA1 orBRCA2 genes are at increased risk of breast cancer.

[0248] XII. Methods of Inhibiting an Immune Response AgainstRepro-EN-1.0 or IB1

[0249] Women with endometriosis exhibit auto-antibodies againstRepro-EN-1.0 or IB1. This fact supports the idea that endometriosisinvolves an auto-immune response. Thus, this invention provides methodsuseful for inhibiting an immune response against Repro-EN-1.0 or IB1. Inone embodiment, the methods include suppressing the immune system inpersons with endometriosis. This includes, for example, theadministration of immunosuppressive drugs such as anti-histamines,anti-inflammatories such as steroids, or cyclosporin or anti-idiotypicantibodies that recognize auto-antibodies against Repro-EN-1.0 or IB1.

[0250] In one embodiment of the invention, the immune response can bediminished by the eliciting in the subject anti-idiotypic antibodiesagainst auto-antibodies that specifically recognize Repro-EN-1.0 or IB1.Anti-idiotypic antibodies are produced by immunizing the subject withantibodies against Repro-EN-1.0 or IB1. The amount of delivery to elicitan immune response can be about 1 μg to about 500 μg given with one ormore booster administrations over about six weeks. Anti-idiotypicantibodies bind to the antigen binding site of the Repro-EN-1.0 or IB1antibodies, thereby blocking their function.

[0251] In another embodiment of the invention the method involvesinducing anergy in T cells involved in a humoral or cell-mediated immuneresponse against Repro-EN-1.0 or IB1. Anergy can be induced byadministering to a subject an MHC-peptide complex that comprises an MHCmolecule coupled to a peptide epitope from Repro-EN-1.0 or IB1.

[0252] MHC Class I and MHC Class II molecules bind peptides havingparticular amino acid motifs in binding pockets located at theamino-terminus of the molecules. The MHC Class II molecule is a dimerformed from an alpha and a beta chain. The binding pocket is formed fromportions of both chains. However, a single beta chain suffices to bind apeptide having the appropriate amino acid motif.

[0253] MHC-peptide complexes are formed by contacting a peptide havingthe appropriate motif with an isolated MHC Class II molecule.Alternatively, the complexes can be formed by creating fusion proteinscontaining both the MHC molecule and the polypeptide epitope.

[0254] Methods of inducing anergy involve administering an isolatedMHC-peptide complex to an individual suffering from the auto-immunedisease. Isolated complexes are complexes that do not exist anchoredonto a cell surface. MHC-peptide complexes and methods of using them toinduce anergy are described in, for example, U.S. Pat. Nos. 5,468,481(S. D. Sharma et al.) and 5,734,023 (B. Nag et al.).

[0255] MHC Class II molecules bind peptides having particular amino acidmotifs well known in the art. HLA-A1 binding motif includes a firstconserved residue of T, S or M, a second conserved residue of D or E,and a third conserved residue of Y. Other second conserved residues areA, S or T. The first and second conserved residues are adjacent and arepreferably separated from the third conserved residue by 6 to 7residues. A second motif consists of a first conserved residue of E or Dand a second conserved residue of Y where the first and second conservedresidues are separated by 5 to 6 residues. The HLA-A3.2 binding motifincludes a first conserved residue of L, M, I, V, S, A, T and F atposition 2 and a second conserved residue of K, R or Y at the C-terminalend. Other first conserved residues are C, G or D and alternatively E.Other second conserved residues are H or F. The first and secondconserved residues are preferably separated by 6 to 7 residues. TheHLA-AL 1 binding motif includes a first conserved residue of T or V atposition 2 and a C-terminal conserved residue of K. The first and secondconserved residues are preferably separated by 6 or 7 residues. TheHLA-A24.1 binding motif includes a first conserved residue of Y, F or Wat position 2 and a C terminal conserved residue of F, I, W, M or L. Thefirst and second conserved residues are preferably separated by 6 to 7residues.

[0256] This invention also provides a peptide comprising a linearepitope derived from the Repro-EN-1.0 or IB1, which specifically bindsto an MHC molecule. In certain embodiments, the peptide has between 8and 12 amino acids and the linear epitope has a Class I MHC moleculebinding motif.

[0257] The following chart provides portions of the amino acid sequenceof Repro-EN-1.0 (SEQ ID NO:2). Amino acid numbers are indicated.Bracketed bars over the amino acid sequence indicate vertebrate MHCClass I or MHC Class II binding motifs. Amino acid numbers areindicated. Peptides of about 8-15 amino acids in length that includethese motifs, including peptides whose entire amino acid sequence isselected from the sequence of Repro-EN-1.0, bind to MHC molecules andcan be used to induce a cell-mediated or humoral immune response againstRepro-EN-1.0. Most of the sequence of Repro-EN-1.0 is exposed on theprotein surface and is capable of eliciting an immune response.

[0258] This invention also provides a pharmaceutical composition capableof inducing anergy against Repro-EN-1.0 comprising a pharmaceuticallyacceptable carrier and an effective amount of an MHC-peptide complex ofthis invention. The complex is capable of inducing anergy in Class IMHC-restricted cytotoxic T-lymphocytes or Class II MHC-restricted immuneresponse against cells expressing Repro-EN-1.0.

[0259] Repro-EN-1.0 includes many amino acid binding motifs for MHCClass I and MHC Class II. These motifs are provided in Table 1. Theamino acid sequence numbers are provided and the motifs are indicatedwith bars. TABLE 1               |--|           |--|       |--| 337pnvslmqrmsdmlsrwfeeasevaqsnrgrgrsrp (SEQ ID NO:11)           347350    358 361                351 354        |--| 386vpsspdlevsetamevdtpaeqflq (SEQ ID NO:12)        396 399           |--|475 pvlslhystegtttstiklnftdew (SEQ ID NO:13)           485 489          |--| 536 etkapeessedvtkyqeqvsaenp (SEQ ID NO:14)           546549           |--| 561 enhinitqsdkftakpldsnsgern (SEQ ID NO:15)          571 574           |--| 624 ntnpepqfqteatgpsaheetstr (SEQ IDNO:16)           634 636          |--|           |--|      |----|    |---| 675drrsavariqeffrrrkerkemeeldtlnirrplvkmvykghrnsrtmikeanfqganfv (SEQ IDNO:17)           685 688        700 703   710  714 720 724          |--|         |---| 739 dcghifiwdrhtaehlmlleadnhvvnclqphpfdpi(SEQ ID NO:18)           749 752      762 765            |---|    |--||--||---||---|             |---| 776lassgidydikiwspleesrifnrkladevitrnelmleetrntitvpasfmlrmlasln (SEQ IDNO:19)             786 789  795 798  804 808                  829 833                        800 803   810 814      |---| sgqenenedee (SEQ IDNO:20)      855858

[0260] XIII. Transgenic Non-Human Animals

[0261] This invention also provides non-human mammals transgenic forRepro-EN-1.0 and IB1. As used herein, “animal transgenic forRepro-EN-1.0 or IB1” refers to an animal, in particular a mammal, whosegerm cells (i.e., oocytes or sperm), at least, comprise a recombinantnucleic acid molecule comprising expression control sequencesoperatively linked to a nucleic acid sequence encoding Repro-EN-1.0 orIB1. Such animals are useful, for example, as models in the study ofendometriosis, spontaneous abortion and disease pathways.

[0262] In one embodiment, the expression control sequences are notnaturally found operatively linked to Repro-EN-1.0. In one embodiment,the recombinant nucleic acid comprises a non-native Repro-EN-1.0 codingsequence, i.e., a Repro-EN-1.0 sequence that the species does notproduce in nature. In one embodiment, the Repro-EN-1.0 is a humanRepro-EN-1.0. In another embodiment, the expression control sequencesare non-native expression control sequences introduced into the germcells so as to recombine with the naturally occurring gene and controlits expression. Particularly useful transgenic mammals of this inventioninclude rabbits and rodents such as mice.

[0263] The transgenic animals of this invention are produced, forexample, by introducing the recombinant nucleic acid molecule into afertilized egg or embryonic stem (ES) cell, typically by microinjection,electroporation, lipofection, particle-mediated gene transfer. Thetransgenic animals express the heterologous nucleotide sequence intissues depending upon whether the promoter is inducible by a signal tothe cell, or is constitutive. Transgenic animals can be bred withnon-transgenic animals to produce transgenic animals with mixedcharacteristics.

[0264] XIV. Methods for Screening for Compounds That Regulate Expressionof Repro-EN-1.0 or IB1

[0265] Compounds that regulate the expression of Repro-EN-1.0 and IB1are candidates as therapeutic agents in the treatment of breast, uterineor prostate cancer. This invention provides methods for determiningwhether a compound regulates (e.g., activates or inhibits) expression ofRepro-EN-1.0 or IB1.

[0266] Methods for determining whether a compound regulates Repro-EN-1.0or IB1 expression involve administering to a cell or a test animalhaving an expressible Repro-EN-1.0 or IB1 gene with the compound, anddetermining whether expression Repro-EN-1.0 or IB1 is altered. In oneembodiment, the methods involve administering the compound to a culturecomprising the cell or to a test animal that has cells expressingRepro-EN-1.0 or IB1, measuring the amount of the Repro-EN-1.0 or IB1polynucleotide or polypeptide in a sample from the culture or theanimal, and determining whether the measured amount is different thanthe amount in a sample from the culture or from the animal under controlconditions (e.g., to which no compound has been administered).Statistically significant (p<0.05) differences between the amountmeasured from the test sample and from the control sample are recordedand indicate that the compound alters the amount of Repro-EN-1.0 or IB1produced by the cell.

[0267] The compound to be tested can be selected from a number ofsources. For example, combinatorial libraries of molecules are availablefor screening experiments. Using such libraries, thousands of moleculescan be screened for regulatory activity. In one preferred embodiment,high throughput screening methods involve providing a library containinga large number of potential therapeutic compounds (candidate compounds).Such “combinatorial chemical libraries” are then screened in one or moreassays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds” or can themselves be used as potential oractual therapeutics.

[0268] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to: peptoids (PCT Publication No WO 91/19735, Dec. 26,1991), encoded peptides (PCT Publication WO 93/20242, Oct. 14, 1993),random bio-oligomers (PCT Publication WO 92/00091, Jan. 9, 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries, peptide nucleic acid libraries (see, e.g., U.S. Pat. No.5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996) NatureBiotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al. (1996) Science, 274: 1520-1522, andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum (1993) C&EN, January 18, page 33,isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones and metathiazanonesU.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat. Nos. 5,525,735 and5,519,134, morpholino compounds U.S. Pat. No. 5,506,337, benzodiazepinesU.S. Pat. No. 5,288,514, and the like).

[0269] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).

[0270] In one embodiment this invention provides inhibitory compoundsthat inhibit expression of Repro-EN-1.0 or IB1 identified oridentifiable by the screening methods of this invention.

[0271] XV. Genomics

[0272] The identification of cognate or polymorphic forms of theRepro-EN-1.0 or IB1 gene and the tracking of those polymorphisms inindividuals and families is important in genetic screening. Accordingly,this invention provides methods useful in detecting polymorphic forms ofthe Repro-EN-1.0 or IB1 gene. The methods involve comparing the identityof a nucleotide or amino acid at a selected position within the sequenceof a test Repro-EN-1.0 or IB1 gene with the nucleotide or amino acid atthe corresponding position from the sequence of native Repro-EN-1.0 (SEQID NO:1) or IB1 (SEQ ID NO:3). The comparison can be carried out by anymethods known in the art, including direct sequence comparison bynucleotide sequencing, sequence comparison or determination byhybridization or identification of RFLPs.

[0273] In one embodiment, the method involves nucleotide or amino acidsequencing of the entire test polynucleotide or polypeptide, or asubsequence from it, and comparing that sequence with the sequence ofnative Repro-EN-1.0 or IB1. In another embodiment, the method involvesidentifying restriction fragments produced upon restriction enzymedigestion of the test polynucleotide and comparing those fragments withfragments produced by restriction enzyme digestion of nativeRepro-EN-1.0 or IB1 gene. Restriction fragments from the native gene canbe identified by analysis of the sequence to identify restriction sites.Another embodiment involves the use of oligonucleotide arrays. (See,e.g., Fodor et al., U.S. Pat. No. 5,445,934.) The method involvesproviding an oligonucleotide array comprising a set of oligonucleotideprobes that define sequences selected from the native Repro-EN-1.0 orIB1 sequence, generating hybridization data by performing ahybridization reaction between the target polynucleotide molecules andthe probes in the set and detecting hybridization between the targetmolecules and each of the probes in the set and processing thehybridization data to determine nucleotide positions at which theidentity of the target molecule differs from that of native Repro-EN-1.0or IB1. The comparison can be done manually, but is more convenientlydone by a programmable, digital computer.

[0274] The following examples are offered by way of illustration, not byway of limitation.

EXAMPLES

[0275] I. Construction of a Human Endometrial Carcinoma Cell Line(RL95-2) cDNA Expression Library

[0276] A. Material and Methods

[0277] Poly A+ RNA isolated from RL95-2, was used as a template forfirst strand cDNA synthesis. The poly A+ RNA was analyzed by denaturinggel electrophoresis and ranged in size from 0.2 to 10 Kb (10). An oligodT primer containing an internal, protected XhoI site was annealed inthe presence of a nucleotide mixture containing 5-methyl dCTP, andextended with MMTV reverse transcriptase.

[0278] Second strand synthesis of the RL95-2 cDNA/RNA hybrid wascompleted by the addition of RNase H, DNA polymerase 1, and dNTPs to thefirst strand synthesis reaction. Pfu DNA polymerase was used toblunt-end the double stranded RL95-2 cDNA followed by the ligation ofEcoRl adapters. The cDNA was kinased and digested with Xhol and EcoRlbefore size fractionation on Sephacryl S-500 columns. The sizefractionated cDNA was recovered and the quantified on ethidium bromidecontaining plates against a set of serially-diluted DNA standards. ThecDNA contained in the first two column fractionations was directionallyligated, in the sense orientation, to Xhol/EcoRl-digested uniZAP phagevector arms. Initially, approximately 25 ng (per fraction) of the cDNAwas ligated and packaged into bacteriophage particles. Subsequently, theapproximately 100 ng remaining cDNA in fractions 1 and 2 was packagedinto bacteriophage particles using several reactions of the lambda phagepackaging extract (Stratagene).

[0279] After packaging, the primary human RL95-2 library was titered byinfection of the XL1 Blue host strain. The ratio ofrecombinant:nonrecombinant phage was determined by plating infected XL1Blue in the presence of IPTG and XGal. The number of blue(non-recombinant) or white (recombinant) plaques were quantified using aManostat colony counter. Ninety-eight and one-half (98.5) percent of thephage in the human RL95-2 cDNA library were recombinant and the primaryphage library base consisted of 2.2×10⁶ independent clones. The humanRL95-2 cDNA library was amplified once and re-titered as above. Thetiter of the amplified library was 3×10⁹ pfu/ml.

[0280] The average size of the cDNA inserts was determined by PCRamplification. Twenty well-isolated phage plaques were cored and thecDNA inserts were amplified using T7/T3 specific oligonucleotides whichhybridize to sites flanking the cDNA insertion site contained within thelambda phage vector. The PCR products were analyzed by agarose gelelectrophoresis and visualized by ethidium bromide staining. Ninety—fivepercent of the cored plaques contained inserts varying in size from 0.5to 2.5 Kb with an average size of 1.5 Kb.

[0281] II. Library Screening

[0282] To identify endometrial autoantigens that could be used todevelop an endometriosis immunoassay, pooled sera from patients (n=17)with laparoscopy confirmed endometriosis was used as a probe to screenthe RL95-2 cDNA expression library using the following methods.

[0283] A. Preadsorption of Serum.

[0284] Antibodies that react with expression library host strains wereimmunoadsorbed from patient serum by using BNN97 and Y1090 E. colilysate-conjugated sepharose beads (5′→3′) following the manufacturer'sprotocols. Briefly, 2 ml of host stain lysate-conjugated sepharose beadswere washed twice with sterile Tris buffered saline (TBS). The beadswere resuspended in 4 mls of serum diluted ½ in TBS. Following a 16 hourincubation at 4° C., the sepharose beads were collected bycentrifugation at 1,000× g for 2 min. The supernatant was removed andthe beads were washed with 4 mls of sterile TBS. After centrifugation at1,000× g for 2 min, the supernatants were collected, pooled and used toscreen the RL95-2-specific cDNA expression library.

[0285] B. Screening the RL95-2 cDNA Library.

[0286] Approximately 106 infectious phage particles were incubated withXL-1 blue host cells and plated at density of 50,000 phage per 150 mmdish using standard protocols (Stratagene). After incubating for 5 hoursat 42° C., the phage plaques were overlaid with nitrocellulose membranes(Protran; Schleicher & Schuell) that had been soaked in a 10 mMisopropyl-1-thio-b-D—galactopyranoside (IPTG) solution. Following a 4hour incubation at 37° C., the membranes were removed and washed threetimes for 15 min in TBS with 0.05% Tween₂₀ (TTBS). The membranes wereincubated with blocking solution (1% Bovine serum albumin [fraction V]in TBS) for 1 hour at room temperature prior to a 1 hour incubation withpreadsorbed patient serum diluted {fraction (1/10)} (final dilution of{fraction (1/40)}) in blocking solution. The membranes were washed threetime for 15 min with TTBS prior to the addition of alkalinephosphatase-conjugated goat anti-human Ig(G,A,M) (Pierce) diluted{fraction (1/25,000)} in blocking solution. After a 1 hour incubation atroom temperature the membranes were washed three times with TTBS asdescribed above and once with TBS. The membranes were incubated withenzyme substrate (Western Blue; Promega) for approximately 30 min andthe enzymatic reaction was terminated by briefly incubating themembranes with stop solution (Tris-HCl pH 2.9; 1 mM EDTA). Severalimmunoreactive phage plaques were selected and transferred to 500 μl ofSM buffer (100 mM NaCl, 8 mM MgSO₄, 50 mM Tris-HCl pH 7.5, 0.01%gelatin) containing 20 μl of chloroform. The selected phage were elutedfrom the agar and plated at a density of approximately 1,000 phage per100 mm dish and screen as described above. To insure that the selectedphage plaque, named Repro-EN-1.0, represented a single clone thescreening process was repeated a third time as described above.

[0287] C. Excision of Repro-EN-1.0 Phagemid.

[0288] Plasmid containing the cDNA insert for Repro-EN-1.0 was excisedfrom the phage clone using the manufacturer's protocols (Stratagene).The size of the Repro-EN-1.0 insert was determined by releasing the cDNAfragment from the rescued pBluescript/Repro-EN-1.0 plasmid with therestriction enzymes EcoRl and Xhol. The released insert was sizefractionated by agarose-gel electrophoresis and the apparent length ofthe insert was determined by comparing its migration position with a DNAstandard (1 kb ladder; Gibco BRL). The insert migrated at approximately2.0 Kb.

[0289] D. Identification of the IB1 Clone.

[0290] An alternately spliced variant of Repro-EN-1.0. A commercialhuman heart cDNA library (Clontech) was screened with a radiolabeledprobe mapping within the amino terminus of the Repro-EN-1.0 codingsequence (nt 203 to 897). One of the two clones isolated contained acDNA insert of 3.4 Kb which possessed an extra 231 base pair insertwithin the Repro-EN-1.0 coding sequence.

[0291] III. Characterization

[0292] A. Sequence Analysis

[0293] The nucleotide sequence of Repro-EN-1.0 was determined by using amodified protocol of the dideoxy chain termination method of Sanger etal. and USB Sequenase 2.0 (Barker, D. F. 1993, Biotechniques). The aminoacid sequence was predicted using the Intelligenetic TRANSLATE programand sequence homologies were determined with BLAST data base searchalgorithms. The deduced amino acid sequence (in the expected frame for afusion protein) was novel.

[0294] B. Tissue Expression Analysis

[0295] The Repro-EN-1.0 expression distribution was determined byNorthern blot analysis using poly A+ RNA collected from human spleen,thymus, prostate, testis, ovary, small intestine, colon, and peripheralblood leukocytes (MTN human blot 11; Clontech) and human heart, brain,placenta, lung, liver, skeletal muscle, kidney and pancreas (MTN humanblot 1-Clontech) and the manufacturer' suggested protocols (Clontech).The immobilized poly A+ RNA samples were incubated with a random primelabeled probe that represented the Repro-EN-1.0 insert. The probe wasgenerated by using and EcoRl/Xhol released fragment of pRepro-EN-1.0 astemplate for a random prime labeled reaction as described in themanufacturer's manual (Megaprime kit; Amersham). The Northern blotmembranes were prehybridized with 6 mls of ExpressHyb solution(Clontech) followed by a 1 hour incubation at 68° C. with approximately0.5 ng of radiolabeled probe (approx. 2×105 cpm) in 5 mls of ExpressHybsolution. Unbound probe was removed by washing the membranes three timeswith 2× SSC, 0.05% SDS at room temperature for 10 min for each washfollowed by twice with 0.1× SSC, 0.1% SDS at 50° C. for 15 min for eachwash. The apparent length of RNA species, a 3.4 Kb mRNA, and tissuedistribution was determined by autoradiography. Expression was detectedprimarily in skeletal muscle, heart and testis; and to a lesser extentin other tissues, but was not detected in lung or peripheral bloodmononuclear cells (PBMC). Expression of Repro-EN-1.0 was up-regulated inbreast and uterine carcinomas relative to their normal counterparts, washighly expressed in both normal fallopian tube and fallopian tubecarcinoma, and was expressed at low levels in both normal ovary andovarian carcinoma (FIG. 2 and FIG. 3). Expression of Repro-EN-1.0 inRL95-2 (endometrium carcinoma) cells is lower than in LNCaP (humanprostate adenocarcinoma), PC-12 (rat cell line) and BT12 (human breastcarcinoma cell line) cells and undetectable in a mouse hybridoma cellline (3E10; negative control) (FIG. 4). In addition, expression innormal endometrium is undetectable.

[0296] C. Homologue Analysis

[0297] To determine the level of nucleotide conservation of Repro-EN-1.0in different species, a Southern blot analysis using EcoRl digestedgenomic DNA collected from human, monkey, rat, mouse, dog, cow, rabbit,chicken and yeast was performed as described in the manufacture's manual(ZooBlot; Clontech). Briefly, the immobilized EcoRl digested genomic DNAsamples were prehybridized with 6 mls of ExpressHyb (Clontech) andincubated for 1 hour at 68° C. with 1.0 ng (approx. 4×10⁵ cpm in 5 mls)of a random prime labeled probe that represented the Repro-EN-1.0insert. Unbound probe was removed by washing the membranes once with 2×SSC, 0.05% SDS at room temperature for 30 min followed by one wash with0.1× SSC, 0.1% SDS at 50C for 30 min. Identification of homologues indifferent species was determine by autoradiography. (See FIG. 4) Thesequence is highly conserved between human and non-human primates(Monkey).

[0298] IV. Antibodies

[0299] Antibodies to peptides of the clone and/or to recombinant proteinwere generated in rabbits. This antisera was used to develop aRepro-EN-1.0 ELISA.

[0300] V. Recombinant Protein

[0301] The predicted ORF for Repro-EN-1 was subcloned into an expressionvector, and the recombinant protein was expressed and purified byNi—Chelate chromatography using standard methodologies.

[0302] VI. Elisa

[0303] The purified recombinant Repro-EN-1 protein was used as a targetantigen in an ELISA (EndX™ ELISA) designed to detect antigen-specificautoantibodies in patient serum.

[0304] The present invention provides a novel nucleotide sequenceencoding Repro-EN-1.0, Repro-EN-1.0 polypeptides, IB1, IB1 polypeptidesand methods of using these materials. While specific examples have beenprovided, the above description is illustrative and not restrictive.Many variations of the invention will become apparent to those skilledin the art upon review of this specification. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

[0305] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument Applicants do not admit that any particular reference is “priorart” to their invention.

1 20 1 3189 DNA Homo sapiens CDS (176)..(2755) 1 cggccgggct tcaggggcccaggcgccgct gctgccaccg ccatctaacg ctgcgccctg 60 gaggcccggc gcgcggatggtgccggtgcg gctcgggtgt tgaaacgggt gtcccctccc 120 cctcctcccc tcccccacgcggtggtctcc cctcccaccc ggctcaggca gagcc atg 178 Met 1 tct cgg ggt ggc tcctac cca cac ctg ttg tgg gac gtg agg aaa agg 226 Ser Arg Gly Gly Ser TyrPro His Leu Leu Trp Asp Val Arg Lys Arg 5 10 15 ttc ctc ggg ctg gag gacccg tcc cgg ctg cgg agt cgc tac ctg gga 274 Phe Leu Gly Leu Glu Asp ProSer Arg Leu Arg Ser Arg Tyr Leu Gly 20 25 30 aga aga gaa ttt atc caa agatta aaa ctt gaa gca acc ctt aat gtg 322 Arg Arg Glu Phe Ile Gln Arg LeuLys Leu Glu Ala Thr Leu Asn Val 35 40 45 cat gat ggt tgt gtt aat aca atctgt tgg aat gac act gga gaa tat 370 His Asp Gly Cys Val Asn Thr Ile CysTrp Asn Asp Thr Gly Glu Tyr 50 55 60 65 att tta tct ggc tca gat gac accaaa tta gta att agt aat cct tac 418 Ile Leu Ser Gly Ser Asp Asp Thr LysLeu Val Ile Ser Asn Pro Tyr 70 75 80 agc aga aag gtt ttg aca aca att cgttca ggg cac cga gca aac ata 466 Ser Arg Lys Val Leu Thr Thr Ile Arg SerGly His Arg Ala Asn Ile 85 90 95 ttt agt gca aag ttc tta cct tgt aca aatgat aaa cag att gta tcc 514 Phe Ser Ala Lys Phe Leu Pro Cys Thr Asn AspLys Gln Ile Val Ser 100 105 110 tgc tct gga gat gga gta ata ttt tat accaac gtt gag caa gat gca 562 Cys Ser Gly Asp Gly Val Ile Phe Tyr Thr AsnVal Glu Gln Asp Ala 115 120 125 gaa acc aac aga caa tgc caa ttt acg tgtcat tat gga act act tat 610 Glu Thr Asn Arg Gln Cys Gln Phe Thr Cys HisTyr Gly Thr Thr Tyr 130 135 140 145 gag att atg act gta ccc aat gac ccttac act ttt ctc tct tgt ggt 658 Glu Ile Met Thr Val Pro Asn Asp Pro TyrThr Phe Leu Ser Cys Gly 150 155 160 gaa gat gga act gtt agg tgg ttt gataca cgc atc aaa act agc tgc 706 Glu Asp Gly Thr Val Arg Trp Phe Asp ThrArg Ile Lys Thr Ser Cys 165 170 175 aca aaa gaa gat tgt aaa gat gat atttta att aac tgt cga cgt gct 754 Thr Lys Glu Asp Cys Lys Asp Asp Ile LeuIle Asn Cys Arg Arg Ala 180 185 190 gcc acg tct gtt gct att tgc cca ccaata cca tat tac ctt gct gtt 802 Ala Thr Ser Val Ala Ile Cys Pro Pro IlePro Tyr Tyr Leu Ala Val 195 200 205 ggt tgt tct gac agc tca gta cga atatat gat cgg cga atg ctg ggc 850 Gly Cys Ser Asp Ser Ser Val Arg Ile TyrAsp Arg Arg Met Leu Gly 210 215 220 225 aca aga gct aca ggg aat tat gcaggt cga ggg act act gga atg gtt 898 Thr Arg Ala Thr Gly Asn Tyr Ala GlyArg Gly Thr Thr Gly Met Val 230 235 240 gcc cgt ttt att cct tcc cat cttaat aat aag tcc tgc aga gtg aca 946 Ala Arg Phe Ile Pro Ser His Leu AsnAsn Lys Ser Cys Arg Val Thr 245 250 255 tct ctg tgt tac agt gaa gat ggtcaa gag att ctc gtt agt tac tct 994 Ser Leu Cys Tyr Ser Glu Asp Gly GlnGlu Ile Leu Val Ser Tyr Ser 260 265 270 tca gat tac ata tat ctt ttt gacccg aaa gat gat aca gca cga gaa 1042 Ser Asp Tyr Ile Tyr Leu Phe Asp ProLys Asp Asp Thr Ala Arg Glu 275 280 285 ctt aaa act cct tct gcg gaa gagaga aga gaa gag ttg cga caa cca 1090 Leu Lys Thr Pro Ser Ala Glu Glu ArgArg Glu Glu Leu Arg Gln Pro 290 295 300 305 cca gtt aag cgt ttg aga cttcgt ggt gat tgg tca gat act gga ccc 1138 Pro Val Lys Arg Leu Arg Leu ArgGly Asp Trp Ser Asp Thr Gly Pro 310 315 320 aga gca agg ccg gag agt gaacga gaa cga gat gga gag cag agt ccc 1186 Arg Ala Arg Pro Glu Ser Glu ArgGlu Arg Asp Gly Glu Gln Ser Pro 325 330 335 aat gtg tca ttg atg cag agaatg tct gat atg tta tca aga tgg ttt 1234 Asn Val Ser Leu Met Gln Arg MetSer Asp Met Leu Ser Arg Trp Phe 340 345 350 gaa gaa gca agt gag gtt gcacaa agc aat aga gga cga gga aga tct 1282 Glu Glu Ala Ser Glu Val Ala GlnSer Asn Arg Gly Arg Gly Arg Ser 355 360 365 cga ccc aga ggt gga aca agtcaa tca gat att tca act ctt cct acg 1330 Arg Pro Arg Gly Gly Thr Ser GlnSer Asp Ile Ser Thr Leu Pro Thr 370 375 380 385 gtc cca tca agt cct gatttg gaa gtg agt gaa act gca atg gaa gta 1378 Val Pro Ser Ser Pro Asp LeuGlu Val Ser Glu Thr Ala Met Glu Val 390 395 400 gat act cca gct gaa caattt ctt cag cct tct aca tcc tct aca atg 1426 Asp Thr Pro Ala Glu Gln PheLeu Gln Pro Ser Thr Ser Ser Thr Met 405 410 415 tca gct cag gct cat tcgaca tca tct ccc aca gaa agc cct cat tct 1474 Ser Ala Gln Ala His Ser ThrSer Ser Pro Thr Glu Ser Pro His Ser 420 425 430 act cct ttg cta tct tctcca gat agt gaa caa agg cag tct gtt gag 1522 Thr Pro Leu Leu Ser Ser ProAsp Ser Glu Gln Arg Gln Ser Val Glu 435 440 445 gca tct gga cac cac acacat cat cag tct gat aac aat aat gaa aag 1570 Ala Ser Gly His His Thr HisHis Gln Ser Asp Asn Asn Asn Glu Lys 450 455 460 465 ctg agc ccc aaa ccaggg aca ggt gaa cca gtt tta agt ttg cac tac 1618 Leu Ser Pro Lys Pro GlyThr Gly Glu Pro Val Leu Ser Leu His Tyr 470 475 480 agc aca gaa gga acaact aca agc aca ata aaa ctg aac ttt aca gat 1666 Ser Thr Glu Gly Thr ThrThr Ser Thr Ile Lys Leu Asn Phe Thr Asp 485 490 495 gaa tgg agc agt atagca tca agt tct aga gga att ggg agc cat tgc 1714 Glu Trp Ser Ser Ile AlaSer Ser Ser Arg Gly Ile Gly Ser His Cys 500 505 510 aaa tct gag ggt caggag gaa tct ttc gtc cca cag agc tca gtg caa 1762 Lys Ser Glu Gly Gln GluGlu Ser Phe Val Pro Gln Ser Ser Val Gln 515 520 525 cca cca gaa gga gacagt gaa aca aaa gct cct gaa gaa tca tca gag 1810 Pro Pro Glu Gly Asp SerGlu Thr Lys Ala Pro Glu Glu Ser Ser Glu 530 535 540 545 gat gtg aca aaatat cag gaa gga gta tct gca gaa aac cca gtt gag 1858 Asp Val Thr Lys TyrGln Glu Gly Val Ser Ala Glu Asn Pro Val Glu 550 555 560 aac cat atc aatata aca caa tca gat aag ttc aca gcc aag cca ttg 1906 Asn His Ile Asn IleThr Gln Ser Asp Lys Phe Thr Ala Lys Pro Leu 565 570 575 gat tcc aac tcagga gaa aga aat gac ctc aat ctt gat cgc tct tgt 1954 Asp Ser Asn Ser GlyGlu Arg Asn Asp Leu Asn Leu Asp Arg Ser Cys 580 585 590 ggg gtt cca gaagaa tct gct tca tct gaa aaa gcc aag gaa cca gaa 2002 Gly Val Pro Glu GluSer Ala Ser Ser Glu Lys Ala Lys Glu Pro Glu 595 600 605 act tca gat cagact agc act gag agt gct acc aat gaa aat aac acc 2050 Thr Ser Asp Gln ThrSer Thr Glu Ser Ala Thr Asn Glu Asn Asn Thr 610 615 620 625 aat cct gagcct cag ttc caa aca gaa gcc act ggg cct tca gct cat 2098 Asn Pro Glu ProGln Phe Gln Thr Glu Ala Thr Gly Pro Ser Ala His 630 635 640 gaa gaa acatcc acc agg gac tct gct ctt cag gac aca gat gac agt 2146 Glu Glu Thr SerThr Arg Asp Ser Ala Leu Gln Asp Thr Asp Asp Ser 645 650 655 gat gat gaccca gtc ctg atc cca ggt gca agg tat cga gca gga cct 2194 Asp Asp Asp ProVal Leu Ile Pro Gly Ala Arg Tyr Arg Ala Gly Pro 660 665 670 ggt gat agacgc tct gct gtt gcc cgt att cag gag ttc ttc aga cgg 2242 Gly Asp Arg ArgSer Ala Val Ala Arg Ile Gln Glu Phe Phe Arg Arg 675 680 685 aga aaa gaaagg aaa gaa atg gaa gaa ttg gat act ttg aac att aga 2290 Arg Lys Glu ArgLys Glu Met Glu Glu Leu Asp Thr Leu Asn Ile Arg 690 695 700 705 agg ccgcta gta aaa atg gtt tat aaa ggc cat cgc aac tcc agg aca 2338 Arg Pro LeuVal Lys Met Val Tyr Lys Gly His Arg Asn Ser Arg Thr 710 715 720 atg ataaaa gaa gcc aat ttc tgg ggt gct aac ttt gta atg act ggt 2386 Met Ile LysGlu Ala Asn Phe Trp Gly Ala Asn Phe Val Met Thr Gly 725 730 735 tct gagtgt ggc cac att ttc atc tgg gat cgg cac act gct gag cat 2434 Ser Glu CysGly His Ile Phe Ile Trp Asp Arg His Thr Ala Glu His 740 745 750 ttg atgctt ctg gaa gct gat aat cat gtg gta aac tgc ctg cag cca 2482 Leu Met LeuLeu Glu Ala Asp Asn His Val Val Asn Cys Leu Gln Pro 755 760 765 cat ccgttt gac cca att tta gcc tca tct ggc ata gat tat gac ata 2530 His Pro PheAsp Pro Ile Leu Ala Ser Ser Gly Ile Asp Tyr Asp Ile 770 775 780 785 aagatc tgg tca cca tta gaa gag tca agg att ttt aac cga aaa ctt 2578 Lys IleTrp Ser Pro Leu Glu Glu Ser Arg Ile Phe Asn Arg Lys Leu 790 795 800 gctgat gaa gtt ata act cga aac gaa ctc atg ctg gaa gaa act aga 2626 Ala AspGlu Val Ile Thr Arg Asn Glu Leu Met Leu Glu Glu Thr Arg 805 810 815 aacacc att aca gtt cca gcc tct ttc atg ttg agg atg ttg gct tca 2674 Asn ThrIle Thr Val Pro Ala Ser Phe Met Leu Arg Met Leu Ala Ser 820 825 830 cttaat cat atc cga gct gac cgg ttg gag ggt gac aga tca gaa ggc 2722 Leu AsnHis Ile Arg Ala Asp Arg Leu Glu Gly Asp Arg Ser Glu Gly 835 840 845 tctggt caa gag aat gaa aat gag gat gag gaa taataaactc tttttggcaa 2775 SerGly Gln Glu Asn Glu Asn Glu Asp Glu Glu 850 855 860 gcacttaaatgttctgaaat ttgtataaga catttattat ttttttttct ttacagagat 2835 ttagtgcaattttaaggtta tggtttttgg agtttttccc tttttttggg ataacctaac 2895 attggtttggaatgattgtg tgcatgaatt tgggagattg tataaaacaa aactagcaga 2955 atgtttttaaaactttttgc cgtgtatgag gagtgctaga aaatgcaaag tgcaatattt 3015 tccctaaccttcaaatgtgg gagcttggat caatgttgaa gaataatttt catcatagtg 3075 aaaatgttggttcaaataaa tttctacact tgccatttgc atgtttgttg ctttctaatt 3135 aaagaaactggttgttttaa gataccctga aaaaaaaaaa aaaaaaaaaa aaaa 3189 2 860 PRT Homosapiens 2 Met Ser Arg Gly Gly Ser Tyr Pro His Leu Leu Trp Asp Val ArgLys 1 5 10 15 Arg Phe Leu Gly Leu Glu Asp Pro Ser Arg Leu Arg Ser ArgTyr Leu 20 25 30 Gly Arg Arg Glu Phe Ile Gln Arg Leu Lys Leu Glu Ala ThrLeu Asn 35 40 45 Val His Asp Gly Cys Val Asn Thr Ile Cys Trp Asn Asp ThrGly Glu 50 55 60 Tyr Ile Leu Ser Gly Ser Asp Asp Thr Lys Leu Val Ile SerAsn Pro 65 70 75 80 Tyr Ser Arg Lys Val Leu Thr Thr Ile Arg Ser Gly HisArg Ala Asn 85 90 95 Ile Phe Ser Ala Lys Phe Leu Pro Cys Thr Asn Asp LysGln Ile Val 100 105 110 Ser Cys Ser Gly Asp Gly Val Ile Phe Tyr Thr AsnVal Glu Gln Asp 115 120 125 Ala Glu Thr Asn Arg Gln Cys Gln Phe Thr CysHis Tyr Gly Thr Thr 130 135 140 Tyr Glu Ile Met Thr Val Pro Asn Asp ProTyr Thr Phe Leu Ser Cys 145 150 155 160 Gly Glu Asp Gly Thr Val Arg TrpPhe Asp Thr Arg Ile Lys Thr Ser 165 170 175 Cys Thr Lys Glu Asp Cys LysAsp Asp Ile Leu Ile Asn Cys Arg Arg 180 185 190 Ala Ala Thr Ser Val AlaIle Cys Pro Pro Ile Pro Tyr Tyr Leu Ala 195 200 205 Val Gly Cys Ser AspSer Ser Val Arg Ile Tyr Asp Arg Arg Met Leu 210 215 220 Gly Thr Arg AlaThr Gly Asn Tyr Ala Gly Arg Gly Thr Thr Gly Met 225 230 235 240 Val AlaArg Phe Ile Pro Ser His Leu Asn Asn Lys Ser Cys Arg Val 245 250 255 ThrSer Leu Cys Tyr Ser Glu Asp Gly Gln Glu Ile Leu Val Ser Tyr 260 265 270Ser Ser Asp Tyr Ile Tyr Leu Phe Asp Pro Lys Asp Asp Thr Ala Arg 275 280285 Glu Leu Lys Thr Pro Ser Ala Glu Glu Arg Arg Glu Glu Leu Arg Gln 290295 300 Pro Pro Val Lys Arg Leu Arg Leu Arg Gly Asp Trp Ser Asp Thr Gly305 310 315 320 Pro Arg Ala Arg Pro Glu Ser Glu Arg Glu Arg Asp Gly GluGln Ser 325 330 335 Pro Asn Val Ser Leu Met Gln Arg Met Ser Asp Met LeuSer Arg Trp 340 345 350 Phe Glu Glu Ala Ser Glu Val Ala Gln Ser Asn ArgGly Arg Gly Arg 355 360 365 Ser Arg Pro Arg Gly Gly Thr Ser Gln Ser AspIle Ser Thr Leu Pro 370 375 380 Thr Val Pro Ser Ser Pro Asp Leu Glu ValSer Glu Thr Ala Met Glu 385 390 395 400 Val Asp Thr Pro Ala Glu Gln PheLeu Gln Pro Ser Thr Ser Ser Thr 405 410 415 Met Ser Ala Gln Ala His SerThr Ser Ser Pro Thr Glu Ser Pro His 420 425 430 Ser Thr Pro Leu Leu SerSer Pro Asp Ser Glu Gln Arg Gln Ser Val 435 440 445 Glu Ala Ser Gly HisHis Thr His His Gln Ser Asp Asn Asn Asn Glu 450 455 460 Lys Leu Ser ProLys Pro Gly Thr Gly Glu Pro Val Leu Ser Leu His 465 470 475 480 Tyr SerThr Glu Gly Thr Thr Thr Ser Thr Ile Lys Leu Asn Phe Thr 485 490 495 AspGlu Trp Ser Ser Ile Ala Ser Ser Ser Arg Gly Ile Gly Ser His 500 505 510Cys Lys Ser Glu Gly Gln Glu Glu Ser Phe Val Pro Gln Ser Ser Val 515 520525 Gln Pro Pro Glu Gly Asp Ser Glu Thr Lys Ala Pro Glu Glu Ser Ser 530535 540 Glu Asp Val Thr Lys Tyr Gln Glu Gly Val Ser Ala Glu Asn Pro Val545 550 555 560 Glu Asn His Ile Asn Ile Thr Gln Ser Asp Lys Phe Thr AlaLys Pro 565 570 575 Leu Asp Ser Asn Ser Gly Glu Arg Asn Asp Leu Asn LeuAsp Arg Ser 580 585 590 Cys Gly Val Pro Glu Glu Ser Ala Ser Ser Glu LysAla Lys Glu Pro 595 600 605 Glu Thr Ser Asp Gln Thr Ser Thr Glu Ser AlaThr Asn Glu Asn Asn 610 615 620 Thr Asn Pro Glu Pro Gln Phe Gln Thr GluAla Thr Gly Pro Ser Ala 625 630 635 640 His Glu Glu Thr Ser Thr Arg AspSer Ala Leu Gln Asp Thr Asp Asp 645 650 655 Ser Asp Asp Asp Pro Val LeuIle Pro Gly Ala Arg Tyr Arg Ala Gly 660 665 670 Pro Gly Asp Arg Arg SerAla Val Ala Arg Ile Gln Glu Phe Phe Arg 675 680 685 Arg Arg Lys Glu ArgLys Glu Met Glu Glu Leu Asp Thr Leu Asn Ile 690 695 700 Arg Arg Pro LeuVal Lys Met Val Tyr Lys Gly His Arg Asn Ser Arg 705 710 715 720 Thr MetIle Lys Glu Ala Asn Phe Trp Gly Ala Asn Phe Val Met Thr 725 730 735 GlySer Glu Cys Gly His Ile Phe Ile Trp Asp Arg His Thr Ala Glu 740 745 750His Leu Met Leu Leu Glu Ala Asp Asn His Val Val Asn Cys Leu Gln 755 760765 Pro His Pro Phe Asp Pro Ile Leu Ala Ser Ser Gly Ile Asp Tyr Asp 770775 780 Ile Lys Ile Trp Ser Pro Leu Glu Glu Ser Arg Ile Phe Asn Arg Lys785 790 795 800 Leu Ala Asp Glu Val Ile Thr Arg Asn Glu Leu Met Leu GluGlu Thr 805 810 815 Arg Asn Thr Ile Thr Val Pro Ala Ser Phe Met Leu ArgMet Leu Ala 820 825 830 Ser Leu Asn His Ile Arg Ala Asp Arg Leu Glu GlyAsp Arg Ser Glu 835 840 845 Gly Ser Gly Gln Glu Asn Glu Asn Glu Asp GluGlu 850 855 860 3 3420 DNA Homo sapiens CDS (176)..(2986) 3 cggccgggcttcaggggccc aggcgccgct gctgccaccg ccatctaacg ctgcgccctg 60 gaggcccggcgcgcggatgg tgccggtgcg gctcgggtgt tgaaacgggt gtcccctccc 120 cctcctcccctcccccacgc ggtggtctcc cctcccaccc ggctcaggca gagcc atg 178 Met 1 tct cggggt ggc tcc tac cca cac ctg ttg tgg gac gtg agg aaa agg 226 Ser Arg GlyGly Ser Tyr Pro His Leu Leu Trp Asp Val Arg Lys Arg 5 10 15 ttc ctc gggctg gag gac ccg tcc cgg ctg cgg agt cgc tac ctg gga 274 Phe Leu Gly LeuGlu Asp Pro Ser Arg Leu Arg Ser Arg Tyr Leu Gly 20 25 30 aga aga gaa tttatc caa aga tta aaa ctt gaa gca acc ctt aat gtg 322 Arg Arg Glu Phe IleGln Arg Leu Lys Leu Glu Ala Thr Leu Asn Val 35 40 45 cat gat ggt tgt gttaat aca atc tgt tgg aat gac act gga gaa tat 370 His Asp Gly Cys Val AsnThr Ile Cys Trp Asn Asp Thr Gly Glu Tyr 50 55 60 65 att tta tct ggc tcagat gac acc aaa tta gta att agt aat cct tac 418 Ile Leu Ser Gly Ser AspAsp Thr Lys Leu Val Ile Ser Asn Pro Tyr 70 75 80 agc aga aag gtt ttg acaaca att cgt tca ggg cac cga gca aac ata 466 Ser Arg Lys Val Leu Thr ThrIle Arg Ser Gly His Arg Ala Asn Ile 85 90 95 ttt agt gca aag ttc tta ccttgt aca aat gat aaa cag att gta tcc 514 Phe Ser Ala Lys Phe Leu Pro CysThr Asn Asp Lys Gln Ile Val Ser 100 105 110 tgc tct gga gat gga gta atattt tat acc aac gtt gag caa gat gca 562 Cys Ser Gly Asp Gly Val Ile PheTyr Thr Asn Val Glu Gln Asp Ala 115 120 125 gaa acc aac aga caa tgc caattt acg tgt cat tat gga act act tat 610 Glu Thr Asn Arg Gln Cys Gln PheThr Cys His Tyr Gly Thr Thr Tyr 130 135 140 145 gag att atg act gta cccaat gac cct tac act ttt ctc tct tgt ggt 658 Glu Ile Met Thr Val Pro AsnAsp Pro Tyr Thr Phe Leu Ser Cys Gly 150 155 160 gaa gat gga act gtt aggtgg ttt gat aca cgc atc aaa act agc tgc 706 Glu Asp Gly Thr Val Arg TrpPhe Asp Thr Arg Ile Lys Thr Ser Cys 165 170 175 aca aaa gaa gat tgt aaagat gat att tta att aac tgt cga cgt gct 754 Thr Lys Glu Asp Cys Lys AspAsp Ile Leu Ile Asn Cys Arg Arg Ala 180 185 190 gcc acg tct gtt gct atttgc cca cca ata cca tat tac ctt gct gtt 802 Ala Thr Ser Val Ala Ile CysPro Pro Ile Pro Tyr Tyr Leu Ala Val 195 200 205 ggt tgt tct gac agc tcagta cga ata tat gat cgg cga atg ctg ggc 850 Gly Cys Ser Asp Ser Ser ValArg Ile Tyr Asp Arg Arg Met Leu Gly 210 215 220 225 aca aga gct aca gggaat tat gca ggt cga ggg act act gga atg gtt 898 Thr Arg Ala Thr Gly AsnTyr Ala Gly Arg Gly Thr Thr Gly Met Val 230 235 240 gcc cgt ttt att ccttcc cat ctt aat aat aag tcc tgc aga gtg aca 946 Ala Arg Phe Ile Pro SerHis Leu Asn Asn Lys Ser Cys Arg Val Thr 245 250 255 tct ctg tgt tac agtgaa gat ggt caa gag att ctc gtt agt tac tct 994 Ser Leu Cys Tyr Ser GluAsp Gly Gln Glu Ile Leu Val Ser Tyr Ser 260 265 270 tca gat tac ata tatctt ttt gac ccg aaa gat gat aca gca cga gaa 1042 Ser Asp Tyr Ile Tyr LeuPhe Asp Pro Lys Asp Asp Thr Ala Arg Glu 275 280 285 ctt aaa act cct tctgcg gaa gag aga aga gaa gag ttg cga caa cca 1090 Leu Lys Thr Pro Ser AlaGlu Glu Arg Arg Glu Glu Leu Arg Gln Pro 290 295 300 305 cca gtt aag cgtttg aga ctt cgt ggt gat tgg tca gat act gga ccc 1138 Pro Val Lys Arg LeuArg Leu Arg Gly Asp Trp Ser Asp Thr Gly Pro 310 315 320 aga gca agg ccggag agt gaa cga gaa cga gat gga gag cag agt ccc 1186 Arg Ala Arg Pro GluSer Glu Arg Glu Arg Asp Gly Glu Gln Ser Pro 325 330 335 aat gtg tca ttgatg cag aga atg tct gat atg tta tca aga tgg ttt 1234 Asn Val Ser Leu MetGln Arg Met Ser Asp Met Leu Ser Arg Trp Phe 340 345 350 gaa gaa gca agtgag gtt gca caa agc aat aga gga cga gga aga tct 1282 Glu Glu Ala Ser GluVal Ala Gln Ser Asn Arg Gly Arg Gly Arg Ser 355 360 365 cga ccc aga ggtgga aca agt caa tca gat att tca act ctt cct acg 1330 Arg Pro Arg Gly GlyThr Ser Gln Ser Asp Ile Ser Thr Leu Pro Thr 370 375 380 385 gtc cca tcaagt cct gat ttg gaa gtg agt gaa act gca atg gaa gta 1378 Val Pro Ser SerPro Asp Leu Glu Val Ser Glu Thr Ala Met Glu Val 390 395 400 gat act ccagct gaa caa ttt ctt cag cct tct aca tcc tct aca atg 1426 Asp Thr Pro AlaGlu Gln Phe Leu Gln Pro Ser Thr Ser Ser Thr Met 405 410 415 tca gct caggct cat tcg aca tca tct ccc aca gaa agc cct cat tct 1474 Ser Ala Gln AlaHis Ser Thr Ser Ser Pro Thr Glu Ser Pro His Ser 420 425 430 act cct ttgcta tct tct cca gat agt gaa caa agg cag tct gtt gag 1522 Thr Pro Leu LeuSer Ser Pro Asp Ser Glu Gln Arg Gln Ser Val Glu 435 440 445 gca tct ggacac cac aca cat cat cag tct gaa ttt tta agg ggg cct 1570 Ala Ser Gly HisHis Thr His His Gln Ser Glu Phe Leu Arg Gly Pro 450 455 460 465 gag atagct ttg ctt cgt aag cgc ctg caa caa ctg agg ctt aag aag 1618 Glu Ile AlaLeu Leu Arg Lys Arg Leu Gln Gln Leu Arg Leu Lys Lys 470 475 480 gct gagcag cag agg cag caa gag cta gct gca cat acc cag caa cag 1666 Ala Glu GlnGln Arg Gln Gln Glu Leu Ala Ala His Thr Gln Gln Gln 485 490 495 cct tccact tct gat cag tct tct cat gag ggc tct tca cag gac cct 1714 Pro Ser ThrSer Asp Gln Ser Ser His Glu Gly Ser Ser Gln Asp Pro 500 505 510 cat gcttca gat tct cct tct tct gtg gtt aac aaa cag ctc gga tcc 1762 His Ala SerAsp Ser Pro Ser Ser Val Val Asn Lys Gln Leu Gly Ser 515 520 525 atg tcactt gac gag caa cag gat aac aat aat gaa aag ctg agc ccc 1810 Met Ser LeuAsp Glu Gln Gln Asp Asn Asn Asn Glu Lys Leu Ser Pro 530 535 540 545 aaacca ggg aca ggt gaa cca gtt tta agt ttg cac tac agc aca gaa 1858 Lys ProGly Thr Gly Glu Pro Val Leu Ser Leu His Tyr Ser Thr Glu 550 555 560 ggaaca act aca agc aca ata aaa ctg aac ttt aca gat gaa tgg agc 1906 Gly ThrThr Thr Ser Thr Ile Lys Leu Asn Phe Thr Asp Glu Trp Ser 565 570 575 agtata gca tca agt tct aga gga att ggg agc cat tgc aaa tct gag 1954 Ser IleAla Ser Ser Ser Arg Gly Ile Gly Ser His Cys Lys Ser Glu 580 585 590 ggtcag gag gaa tct ttc gtc cca cag agc tca gtg caa cca cca gaa 2002 Gly GlnGlu Glu Ser Phe Val Pro Gln Ser Ser Val Gln Pro Pro Glu 595 600 605 ggagac agt gaa aca aaa gct cct gaa gaa tca tca gag gat gtg aca 2050 Gly AspSer Glu Thr Lys Ala Pro Glu Glu Ser Ser Glu Asp Val Thr 610 615 620 625aaa tat cag gaa gga gta tct gca gaa aac cca gtt gag aac cat atc 2098 LysTyr Gln Glu Gly Val Ser Ala Glu Asn Pro Val Glu Asn His Ile 630 635 640aat ata aca caa tca gat aag ttc aca gcc aag cca ttg gat tcc aac 2146 AsnIle Thr Gln Ser Asp Lys Phe Thr Ala Lys Pro Leu Asp Ser Asn 645 650 655tca gga gaa aga aat gac ctc aat ctt gat cgc tct tgt ggg gtt cca 2194 SerGly Glu Arg Asn Asp Leu Asn Leu Asp Arg Ser Cys Gly Val Pro 660 665 670gaa gaa tct gct tca tct gaa aaa gcc aag gaa cca gaa act tca gat 2242 GluGlu Ser Ala Ser Ser Glu Lys Ala Lys Glu Pro Glu Thr Ser Asp 675 680 685cag act agc act gag agt gct acc aat gaa aat aac acc aat cct gag 2290 GlnThr Ser Thr Glu Ser Ala Thr Asn Glu Asn Asn Thr Asn Pro Glu 690 695 700705 cct cag ttc caa aca gaa gcc act ggg cct tca gct cat gaa gaa aca 2338Pro Gln Phe Gln Thr Glu Ala Thr Gly Pro Ser Ala His Glu Glu Thr 710 715720 tcc acc agg gac tct gct ctt cag gac aca gat gac agt gat gat gac 2386Ser Thr Arg Asp Ser Ala Leu Gln Asp Thr Asp Asp Ser Asp Asp Asp 725 730735 cca gtc ctg atc cca ggt gca agg tat cga gca gga cct ggt gat aga 2434Pro Val Leu Ile Pro Gly Ala Arg Tyr Arg Ala Gly Pro Gly Asp Arg 740 745750 cgc tct gct gtt gcc cgt att cag gag ttc ttc aga cgg aga aaa gaa 2482Arg Ser Ala Val Ala Arg Ile Gln Glu Phe Phe Arg Arg Arg Lys Glu 755 760765 agg aaa gaa atg gaa gaa ttg gat act ttg aac att aga agg ccg cta 2530Arg Lys Glu Met Glu Glu Leu Asp Thr Leu Asn Ile Arg Arg Pro Leu 770 775780 785 gta aaa atg gtt tat aaa ggc cat cgc aac tcc agg aca atg ata aaa2578 Val Lys Met Val Tyr Lys Gly His Arg Asn Ser Arg Thr Met Ile Lys 790795 800 gaa gcc aat ttc tgg ggt gct aac ttt gta atg act ggt tct gag tgt2626 Glu Ala Asn Phe Trp Gly Ala Asn Phe Val Met Thr Gly Ser Glu Cys 805810 815 ggc cac att ttc atc tgg gat cgg cac act gct gag cat ttg atg ctt2674 Gly His Ile Phe Ile Trp Asp Arg His Thr Ala Glu His Leu Met Leu 820825 830 ctg gaa gct gat aat cat gtg gta aac tgc ctg cag cca cat ccg ttt2722 Leu Glu Ala Asp Asn His Val Val Asn Cys Leu Gln Pro His Pro Phe 835840 845 gac cca att tta gcc tca tct ggc ata gat tat gac ata aag atc tgg2770 Asp Pro Ile Leu Ala Ser Ser Gly Ile Asp Tyr Asp Ile Lys Ile Trp 850855 860 865 tca cca tta gaa gag tca agg att ttt aac cga aaa ctt gct gatgaa 2818 Ser Pro Leu Glu Glu Ser Arg Ile Phe Asn Arg Lys Leu Ala Asp Glu870 875 880 gtt ata act cga aac gaa ctc atg ctg gaa gaa act aga aac accatt 2866 Val Ile Thr Arg Asn Glu Leu Met Leu Glu Glu Thr Arg Asn Thr Ile885 890 895 aca gtt cca gcc tct ttc atg ttg agg atg ttg gct tca ctt aatcat 2914 Thr Val Pro Ala Ser Phe Met Leu Arg Met Leu Ala Ser Leu Asn His900 905 910 atc cga gct gac cgg ttg gag ggt gac aga tca gaa ggc tct ggtcaa 2962 Ile Arg Ala Asp Arg Leu Glu Gly Asp Arg Ser Glu Gly Ser Gly Gln915 920 925 gag aat gaa aat gag gat gag gaa taataaactc tttttggcaagcacttaaat 3016 Glu Asn Glu Asn Glu Asp Glu Glu 930 935 gttctgaaatttgtataaga catttattat ttttttttct ttacagagat ttagtgcaat 3076 tttaaggttatggtttttgg agtttttccc tttttttggg ataacctaac attggtttgg 3136 aatgattgtgtgcatgaatt tgggagattg tataaaacaa aactagcaga atgtttttaa 3196 aactttttgccgtgtatgag gagtgctaga aaatgcaaag tgcaatattt tccctaacct 3256 tcaaatgtgggagcttggat caatgttgaa gaataatttt catcatagtg aaaatgttgg 3316 ttcaaataaatttctacact tgccatttgc atgtttgttg ctttctaatt aaagaaactg 3376 gttgttttaagataccctga aaaaaaaaaa aaaaaaaaaa aaaa 3420 4 937 PRT Homo sapiens 4 MetSer Arg Gly Gly Ser Tyr Pro His Leu Leu Trp Asp Val Arg Lys 1 5 10 15Arg Phe Leu Gly Leu Glu Asp Pro Ser Arg Leu Arg Ser Arg Tyr Leu 20 25 30Gly Arg Arg Glu Phe Ile Gln Arg Leu Lys Leu Glu Ala Thr Leu Asn 35 40 45Val His Asp Gly Cys Val Asn Thr Ile Cys Trp Asn Asp Thr Gly Glu 50 55 60Tyr Ile Leu Ser Gly Ser Asp Asp Thr Lys Leu Val Ile Ser Asn Pro 65 70 7580 Tyr Ser Arg Lys Val Leu Thr Thr Ile Arg Ser Gly His Arg Ala Asn 85 9095 Ile Phe Ser Ala Lys Phe Leu Pro Cys Thr Asn Asp Lys Gln Ile Val 100105 110 Ser Cys Ser Gly Asp Gly Val Ile Phe Tyr Thr Asn Val Glu Gln Asp115 120 125 Ala Glu Thr Asn Arg Gln Cys Gln Phe Thr Cys His Tyr Gly ThrThr 130 135 140 Tyr Glu Ile Met Thr Val Pro Asn Asp Pro Tyr Thr Phe LeuSer Cys 145 150 155 160 Gly Glu Asp Gly Thr Val Arg Trp Phe Asp Thr ArgIle Lys Thr Ser 165 170 175 Cys Thr Lys Glu Asp Cys Lys Asp Asp Ile LeuIle Asn Cys Arg Arg 180 185 190 Ala Ala Thr Ser Val Ala Ile Cys Pro ProIle Pro Tyr Tyr Leu Ala 195 200 205 Val Gly Cys Ser Asp Ser Ser Val ArgIle Tyr Asp Arg Arg Met Leu 210 215 220 Gly Thr Arg Ala Thr Gly Asn TyrAla Gly Arg Gly Thr Thr Gly Met 225 230 235 240 Val Ala Arg Phe Ile ProSer His Leu Asn Asn Lys Ser Cys Arg Val 245 250 255 Thr Ser Leu Cys TyrSer Glu Asp Gly Gln Glu Ile Leu Val Ser Tyr 260 265 270 Ser Ser Asp TyrIle Tyr Leu Phe Asp Pro Lys Asp Asp Thr Ala Arg 275 280 285 Glu Leu LysThr Pro Ser Ala Glu Glu Arg Arg Glu Glu Leu Arg Gln 290 295 300 Pro ProVal Lys Arg Leu Arg Leu Arg Gly Asp Trp Ser Asp Thr Gly 305 310 315 320Pro Arg Ala Arg Pro Glu Ser Glu Arg Glu Arg Asp Gly Glu Gln Ser 325 330335 Pro Asn Val Ser Leu Met Gln Arg Met Ser Asp Met Leu Ser Arg Trp 340345 350 Phe Glu Glu Ala Ser Glu Val Ala Gln Ser Asn Arg Gly Arg Gly Arg355 360 365 Ser Arg Pro Arg Gly Gly Thr Ser Gln Ser Asp Ile Ser Thr LeuPro 370 375 380 Thr Val Pro Ser Ser Pro Asp Leu Glu Val Ser Glu Thr AlaMet Glu 385 390 395 400 Val Asp Thr Pro Ala Glu Gln Phe Leu Gln Pro SerThr Ser Ser Thr 405 410 415 Met Ser Ala Gln Ala His Ser Thr Ser Ser ProThr Glu Ser Pro His 420 425 430 Ser Thr Pro Leu Leu Ser Ser Pro Asp SerGlu Gln Arg Gln Ser Val 435 440 445 Glu Ala Ser Gly His His Thr His HisGln Ser Glu Phe Leu Arg Gly 450 455 460 Pro Glu Ile Ala Leu Leu Arg LysArg Leu Gln Gln Leu Arg Leu Lys 465 470 475 480 Lys Ala Glu Gln Gln ArgGln Gln Glu Leu Ala Ala His Thr Gln Gln 485 490 495 Gln Pro Ser Thr SerAsp Gln Ser Ser His Glu Gly Ser Ser Gln Asp 500 505 510 Pro His Ala SerAsp Ser Pro Ser Ser Val Val Asn Lys Gln Leu Gly 515 520 525 Ser Met SerLeu Asp Glu Gln Gln Asp Asn Asn Asn Glu Lys Leu Ser 530 535 540 Pro LysPro Gly Thr Gly Glu Pro Val Leu Ser Leu His Tyr Ser Thr 545 550 555 560Glu Gly Thr Thr Thr Ser Thr Ile Lys Leu Asn Phe Thr Asp Glu Trp 565 570575 Ser Ser Ile Ala Ser Ser Ser Arg Gly Ile Gly Ser His Cys Lys Ser 580585 590 Glu Gly Gln Glu Glu Ser Phe Val Pro Gln Ser Ser Val Gln Pro Pro595 600 605 Glu Gly Asp Ser Glu Thr Lys Ala Pro Glu Glu Ser Ser Glu AspVal 610 615 620 Thr Lys Tyr Gln Glu Gly Val Ser Ala Glu Asn Pro Val GluAsn His 625 630 635 640 Ile Asn Ile Thr Gln Ser Asp Lys Phe Thr Ala LysPro Leu Asp Ser 645 650 655 Asn Ser Gly Glu Arg Asn Asp Leu Asn Leu AspArg Ser Cys Gly Val 660 665 670 Pro Glu Glu Ser Ala Ser Ser Glu Lys AlaLys Glu Pro Glu Thr Ser 675 680 685 Asp Gln Thr Ser Thr Glu Ser Ala ThrAsn Glu Asn Asn Thr Asn Pro 690 695 700 Glu Pro Gln Phe Gln Thr Glu AlaThr Gly Pro Ser Ala His Glu Glu 705 710 715 720 Thr Ser Thr Arg Asp SerAla Leu Gln Asp Thr Asp Asp Ser Asp Asp 725 730 735 Asp Pro Val Leu IlePro Gly Ala Arg Tyr Arg Ala Gly Pro Gly Asp 740 745 750 Arg Arg Ser AlaVal Ala Arg Ile Gln Glu Phe Phe Arg Arg Arg Lys 755 760 765 Glu Arg LysGlu Met Glu Glu Leu Asp Thr Leu Asn Ile Arg Arg Pro 770 775 780 Leu ValLys Met Val Tyr Lys Gly His Arg Asn Ser Arg Thr Met Ile 785 790 795 800Lys Glu Ala Asn Phe Trp Gly Ala Asn Phe Val Met Thr Gly Ser Glu 805 810815 Cys Gly His Ile Phe Ile Trp Asp Arg His Thr Ala Glu His Leu Met 820825 830 Leu Leu Glu Ala Asp Asn His Val Val Asn Cys Leu Gln Pro His Pro835 840 845 Phe Asp Pro Ile Leu Ala Ser Ser Gly Ile Asp Tyr Asp Ile LysIle 850 855 860 Trp Ser Pro Leu Glu Glu Ser Arg Ile Phe Asn Arg Lys LeuAla Asp 865 870 875 880 Glu Val Ile Thr Arg Asn Glu Leu Met Leu Glu GluThr Arg Asn Thr 885 890 895 Ile Thr Val Pro Ala Ser Phe Met Leu Arg MetLeu Ala Ser Leu Asn 900 905 910 His Ile Arg Ala Asp Arg Leu Glu Gly AspArg Ser Glu Gly Ser Gly 915 920 925 Gln Glu Asn Glu Asn Glu Asp Glu Glu930 935 5 21 DNA Homo sapiens 5 caggacacag atgacagtga t 21 6 20 DNA Homosapiens 6 agagccttct gatctgtcac 20 7 9 PRT Homo sapiens 7 Lys Thr ProSer Ala Glu Glu Arg Arg 1 5 8 8 PRT Homo sapiens 8 Arg Ala Arg Pro GluSer Glu Arg 1 5 9 7 PRT Homo sapiens 9 Arg Met Ser Asp Met Ser Arg 1 510 9 PRT Homo sapiens 10 Asn Glu Lys Leu Ser Pro Lys Pro Gly 1 5 11 35PRT Homo sapiens 11 Pro Asn Val Ser Leu Met Gln Arg Met Ser Asp Met LeuSer Arg Trp 1 5 10 15 Phe Glu Glu Ala Ser Glu Val Ala Gln Ser Asn ArgGly Arg Gly Arg 20 25 30 Ser Arg Pro 35 12 25 PRT Homo sapiens 12 ValPro Ser Ser Pro Asp Leu Glu Val Ser Glu Thr Ala Met Glu Val 1 5 10 15Asp Thr Pro Ala Glu Gln Phe Leu Gln 20 25 13 25 PRT Homo sapiens 13 ProVal Leu Ser Leu His Tyr Ser Thr Glu Gly Thr Thr Thr Ser Thr 1 5 10 15Ile Lys Leu Asn Phe Thr Asp Glu Trp 20 25 14 24 PRT Homo sapiens 14 GluThr Lys Ala Pro Glu Glu Ser Ser Glu Asp Val Thr Lys Tyr Gln 1 5 10 15Glu Gly Val Ser Ala Glu Asn Pro 20 15 25 PRT Homo sapiens 15 Glu Asn HisIle Asn Ile Thr Gln Ser Asp Lys Phe Thr Ala Lys Pro 1 5 10 15 Leu AspSer Asn Ser Gly Glu Arg Asn 20 25 16 24 PRT Homo sapiens 16 Asn Thr AsnPro Glu Pro Gln Phe Gln Thr Glu Ala Thr Gly Pro Ser 1 5 10 15 Ala HisGlu Glu Thr Ser Thr Arg 20 17 60 PRT Homo sapiens 17 Asp Arg Arg Ser AlaVal Ala Arg Ile Gln Glu Phe Phe Arg Arg Arg 1 5 10 15 Lys Glu Arg LysGlu Met Glu Glu Leu Asp Thr Leu Asn Ile Arg Arg 20 25 30 Pro Leu Val LysMet Val Tyr Lys Gly His Arg Asn Ser Arg Thr Met 35 40 45 Ile Lys Glu AlaAsn Phe Trp Gly Ala Asn Phe Val 50 55 60 18 37 PRT Homo sapiens 18 GluCys Gly His Ile Phe Ile Trp Asp Arg His Thr Ala Glu His Leu 1 5 10 15Met Leu Leu Glu Ala Asp Asn His Val Val Asn Cys Leu Gln Pro His 20 25 30Pro Phe Asp Pro Ile 35 19 60 PRT Homo sapiens 19 Leu Ala Ser Ser Gly IleAsp Tyr Asp Ile Lys Ile Trp Ser Pro Leu 1 5 10 15 Glu Glu Ser Arg IlePhe Asn Arg Lys Leu Ala Asp Glu Val Ile Thr 20 25 30 Arg Asn Glu Leu MetLeu Glu Glu Thr Arg Asn Thr Ile Thr Val Pro 35 40 45 Ala Ser Phe Met LeuArg Met Leu Ala Ser Leu Asn 50 55 60 20 11 PRT Homo sapiens 20 Ser GlyGln Glu Asn Glu Asn Glu Asp Glu Glu 1 5 10

What is claimed is:
 1. A recombinant polynucleotide comprising anucleotide sequence encoding a polypeptide epitope of at least 5 aminoacids of Repro-EN-1.0 (SEQ ID NO:2), wherein the epitope specificallybinds to antibodies from subjects diagnosed with endometriosis.
 2. Thepolynucleotide of claim 1 wherein the nucleotide sequence is selectedfrom the Repro-EN-1.0 sequence of SEQ ID NO:1.
 3. The polynucleotide ofclaim 1 wherein the nucleotide sequence is a native Repro-EN-1.0nucleotide sequence.
 4. The polynucleotide of claim 1 wherein thenucleotide sequence is identical to nucleotides 176 to 2755 of SEQ IDNO:1.
 5. The polynucleotide of claim 1 further comprising an expressioncontrol sequence operatively linked to the nucleotide sequence.
 6. Arecombinant polynucleotide comprising a nucleotide sequence encoding apolypeptide epitope of at least 5 amino acids of IB1 (SEQ ID NO:4),wherein the epitope specifically binds to antibodies from subjectsdiagnosed with endometriosis.
 7. The polynucleotide of claim 6 whereinthe nucleotide sequence is selected from the IB1 sequence of SEQ IDNO:3.
 8. The polynucleotide of claim 6 wherein the nucleotide sequenceis a native IB1 nucleotide sequence.
 9. The polynucleotide of claim 6wherein the nucleotide sequence is identical to nucleotides 176 to 2986of SEQ ID NO:3.
 10. The polynucleotide of claim 6 further comprising anexpression control sequence operatively linked to the nucleotidesequence.
 11. A polynucleotide primer pair which amplifies a nucleotidesequence encoding a polypeptide epitope of at least 5 amino acids ofRepro-EN-1.0, wherein the epitope specifically binds to antibodies fromsubjects diagnosed with endometriosis, the pair comprising: 1) a 3′primer of at least 7 nucleotides that specifically hybridizes to a 3′end of the nucleotide sequence or downstream from the sequence, and 2) a5′ primer of at least 7 nucleotides that specifically hybridizes to the3′ end of the complement of the nucleotide sequence or downstream fromthe complement of the sequence.
 12. The polynucleotide primer pair ofclaim 11 wherein the 3′ primer has a sequence complementary to anucleotide sequence selected from Repro-EN-1.0 cDNA (SEQ ID NO:1), andthe 5′ primer has a sequence identical to nucleotide sequence selectedfrom Repro-EN-1.0 cDNA (SEQ ID NO:1).
 13. The polynucleotide primer pairof claim 11 wherein the pair of polynucleotides are peptide nucleicacids.
 14. A polynucleotide primer pair which amplifies a nucleotidesequence encoding a polypeptide epitope of at least 5 amino acids ofIB1, wherein the epitope specifically binds to antibodies from subjectsdiagnosed with endometriosis, the pair comprising: 1) a 3′ primer of atleast 7 nucleotides that specifically hybridizes to a 3′ end of thenucleotide sequence or downstream from the sequence, and 2) a 5′ primerof at least 7 nucleotides that specifically hybridizes to the 3′ end ofthe complement of the nucleotide sequence or downstream from thecomplement of the sequence.
 15. The polynucleotide primer pair of claim14 wherein the 3′ primer has a sequence complementary to a nucleotidesequence selected from IB1 cDNA (SEQ ID NO:3), and the 5′ primer has asequence identical to nucleotide sequence selected from IB1 cDNA (SEQ IDNO:3).
 16. The polynucleotide primer pair of claim 14 wherein the pairof polynucleotides are peptide nucleic acids.
 17. A recombinant cellcomprising a recombinant polynucleotide comprising an expression controlsequence operatively linked to a nucleotide sequence encoding apolypeptide epitope of at least 5 amino acids of Repro-EN-1.0 (SEQ IDNO:2), wherein the epitope specifically binds to antibodies fromsubjects diagnosed with endometriosis.
 18. A method for detecting atarget polynucleotide comprising a nucleotide sequence selected fromRepro-EN-1.0 cDNA (SEQ ID NO:1) or its complement in a sample comprisingthe steps of: (a) contacting the sample with a polynucleotide probe orprimer comprising a sequence of at least 7 nucleotides that specificallyhybridizes to the nucleotide sequence and (b) detecting whether theprobe or primer has specifically hybridized to the targetpolynucleotide, whereby specific hybridization provides a detection ofthe target polynucleotide in the sample.
 19. The method of claim 18wherein the polynucleotide probe or primer is a peptide nucleic acid.20. A recombinant cell comprising a recombinant polynucleotidecomprising an expression control sequence operatively linked to anucleotide sequence encoding a polypeptide epitope of at least 5 aminoacids of IB1 (SEQ ID NO:4), wherein the epitope specifically binds toantibodies from subjects diagnosed with endometriosis.
 21. A method fordetecting a target polynucleotide comprising a nucleotide sequenceselected from IB1 cDNA (SEQ ID NO:3) or its complement in a samplecomprising the steps of: (a) contacting the sample with a polynucleotideprobe or primer comprising a sequence of at least 7 nucleotides thatspecifically hybridizes to the nucleotide sequence and (b) detectingwhether the probe or primer has specifically hybridized to the targetpolynucleotide, whereby specific hybridization provides a detection ofthe target polynucleotide in the sample.
 22. The method of claim 21wherein the polynucleotide probe or primer is a peptide nucleic acid.23. A purified, recombinant Repro-EN-1.0 polypeptide whose amino acidsequence is identical to that of SEQ ID NO:2, or an allelic variant ofSEQ ID NO:2.
 24. A purified, recombinant IB1 polypeptide whose aminoacid sequence is identical to that of SEQ ID NO:4, or an allelic variantof SEQ ID NO:4.
 25. A purified polypeptide comprising an epitope of atleast 5 amino acids of Repro-EN-1.0 (SEQ ID NO:2), wherein the epitopespecifically binds to antibodies from subjects diagnosed withendometriosis.
 26. A purified polypeptide comprising an epitope of atleast 5 amino acids of IB1 (SEQ ID NO:4), wherein the epitopespecifically binds to antibodies from subjects diagnosed withendometriosis.
 27. A composition consisting essentially of an antibodythat specifically binds to Repro-EN-1.0 polypeptide (SEQ ID NO:2). 28.The composition of claim 27 wherein the antibodies are monoclonalantibodies.
 29. A composition consisting essentially of an antibody thatspecifically binds to IB1 polypeptide (SEQ ID NO:4).
 30. The compositionof claim 27 wherein the antibodies are monoclonal antibodies.
 31. Amethod for detecting a Repro-EN-1.0 polypeptide in a sample, comprisingthe steps of: (a) contacting the sample with an antibody thatspecifically binds to the Repro-EN-1.0 polypeptide and (b) detectingspecific binding between the antibody and Repro-EN-1.0 polypeptide,whereby specific binding provides a detection of Repro-EN-1.0polypeptide in the sample.
 32. A method for detecting a IB1 polypeptidein a sample, comprising the steps of: (a) contacting the sample with anantibody that specifically binds to the IB1 polypeptide and (b)detecting specific binding between the antibody and IB1 polypeptide,whereby specific binding provides a detection of IB1 polypeptide in thesample.
 33. A method for diagnosing endometriosis in a subjectcomprising the steps of: (a) detecting a test amount of an antibody thatspecifically binds to Repro-EN-1.0 polypeptide in a sample from thesubject; and (b) comparing the test amount with a normal range of theantibody in a control sample from a subject who does not suffer fromendometriosis, whereby a test amount above the normal range provides apositive indication in the diagnosis of endometriosis.
 34. The method ofclaim 33 wherein the sample comprises blood serum.
 35. The method ofclaim 33 wherein the antibody is an IgE immunoglobulin.
 36. The methodof claim 33 wherein the antibody is an IgG immunoglobulin.
 37. Themethod of claim 33 wherein the antibody is an IgG₄ immunoglobulin. 38.The method of claim 33 wherein the step of detecting comprises capturingthe antibody from the sample with an immobilized Repro-EN-1.0 or apeptide comprising an epitope of Repro-EN-1.0 and detecting capturedantibody.
 39. The method of claim 33 wherein the step of detectingcomprises capturing the antibody from the sample with an immobilizedanti-immunoglobulin antibody and detecting captured antibody.
 40. Themethod of claim 38 wherein the step of detecting captured antibodycomprises contacting the captured antibody with a detectable antibodythat specifically binds immunoglobulins and detecting binding betweenthe captured antibody and the detectable antibody.
 41. The method ofclaim 39 wherein the step of detecting captured antibody comprisescontacting the captured antibody with Repro-EN-1.0 or a polypeptidecomprising an epitope of Repro-EN-1.0 and detecting binding between thecaptured antibody and the Repro-EN-1.0 or polypeptide.
 42. A method fordiagnosing endometriosis in a subject comprising the steps of: (a)detecting a test amount of an antibody that specifically binds to IB1polypeptide in a sample from the subject; and (b) comparing the testamount with a normal range of the antibody in a control sample from asubject who does not suffer from endometriosis, whereby a test amountabove the normal range provides a positive indication in the diagnosisof endometriosis.
 43. The method of claim 42 wherein the samplecomprises blood serum.
 44. The method of claim 42 wherein the antibodyis an IgE immunoglobulin.
 45. The method of claim 42 wherein theantibody is an IgG immunoglobulin.
 46. The method of claim 42 whereinthe antibody is an IgG₄ immunoglobulin.
 47. The method of claim 42wherein the step of detecting comprises capturing the antibody from thesample with an immobilized IB1 or a peptide comprising an epitope of IB1and detecting captured antibody.
 48. The method of claim 46 wherein thestep of detecting comprises capturing the antibody from the sample withan immobilized anti-immunoglobulin antibody and detecting capturedantibody.
 49. The method of claim 47 wherein the step of detectingcaptured antibody comprises contacting the captured antibody with adetectable antibody that specifically binds immunoglobulins anddetecting binding between the captured antibody and the detectableantibody.
 50. The method of claim 48 wherein the step of detectingcaptured antibody comprises contacting the captured antibody with IB1 ora polypeptide comprising an epitope of IB1 and detecting binding betweenthe captured antibody and the IB1 or polypeptide.
 51. A method for usein following the progress of endometriosis in a subject comprising thesteps of: (a) detecting first and second amounts of an antibody thatspecifically binds Repro-EN-1.0 polypeptide in samples from the subjectat a first and a second time, respectively; and (b) comparing the firstand second amounts, whereby an increase between the first and secondamounts indicates progression of the endometriosis and a decreasebetween the first and second amounts indicates remission of theendometriosis.
 52. A method for use in following the progress ofendometriosis in a subject comprising the steps of: (a) detecting firstand second amounts of an antibody that specifically binds IB1polypeptide in samples from the subject at a first and a second time,respectively; and (b) comparing the first and second amounts, whereby anincrease between the first and second amounts indicates progression ofthe endometriosis and a decrease between the first and second amountsindicates remission of the endometriosis.
 53. An isolated MHC-peptidecomplex comprising: at least a portion of an MHC Class I molecule or anMHC Class II molecule, wherein the portion comprises a binding site thatspecifically binds a peptide having an amino acid binding motif specificto the molecule, and wherein the portion engages in CD4-mediated orCD8-mediated binding to T cells, and a peptide of at least 8 amino acidsin a sequence selected from the amino acid sequence of Repro-EN-1.0 (SEQID NO:2), wherein the peptide comprises the amino acid binding motif andcomprises an epitope that specifically binds to a T cell receptor;wherein the complex specifically binds a T cell having a T cell receptorthat specifically binds to the epitope, and wherein specific bindinginduces anergy in the T cell.
 54. An isolated MHC-peptide complexcomprising: at least a portion of an MHC Class I molecule or an MHCClass II molecule, wherein the portion comprises a binding site thatspecifically binds a peptide having an amino acid binding motif specificto the molecule, and wherein the portion engages in CD4-mediated orCD8-mediated binding to T cells, and a peptide of at least 8 amino acidsin a sequence selected from the amino acid sequence of IB1 (SEQ IDNO:4), wherein the peptide comprises the amino acid binding motif andcomprises an epitope that specifically binds to a T cell receptor;wherein the complex specifically binds a T cell having a T cell receptorthat specifically binds to the epitope, and wherein specific bindinginduces anergy in the T cell.
 55. A method for treating endometriosis ina subject comprising the step of inhibiting an immune response againstRepro-EN-1.0 in the subject.
 56. The method of claim 55 comprisingadministering to the subject an immunosuppressant in an amount effectiveto inhibit the immune response.
 57. The method of claim 55 comprisingadministering to the subject an isolated MHC-peptide complex of claim 54in an amount effective to inhibit the immune response.
 58. The method ofclaim 55 comprising administering to the subject an anti-idiotypicantibody that specifically binds to an antigen binding site of anantibody that specifically binds to Repro-EN-1.0 in an amount effectiveto inhibit the immune response.
 59. A method for treating endometriosisin a subject comprising the step of inhibiting an immune responseagainst IB1 in the subject.
 60. The method of claim 59 comprisingadministering to the subject an immunosuppressant in an amount effectiveto inhibit the immune response.
 61. The method of claim 59 comprisingadministering to the subject an isolated MHC-peptide complex of claim 58in an amount effective to inhibit the immune response.
 62. The method ofclaim 59 comprising administering to the subject an anti-idiotypicantibody that specifically binds to an antigen binding site of anantibody that specifically binds to IB1 in an amount effective toinhibit the immune response.
 63. A screening method for determiningwhether a compound increases or decreases the expression of Repro-EN-1.0in a cell comprising contacting the cell with the compound anddetermining whether the production of Repro-EN-1.0 mRNA or polypeptideare increased or decreased.
 64. A screening method for determiningwhether a compound increases or decreases the expression of IB1 in acell comprising contacting the cell with the compound and determiningwhether the production of IB1 mRNA or polypeptide are increased ordecreased.
 65. A method of detecting a chromosomal translocation of aRepro-EN-1.0 gene comprising the steps of: a) hybridizing a labeledpolynucleotide probe that specifically hybridizes with the Repro-EN-1.0nucleotide sequence of SEQ ID NO:1 or its complement, to a chromosomespread from a cell sample to determine the pattern of hybridization andb) determining whether the pattern of hybridization differs from anormal pattern; whereby a difference in the pattern represents atranslocation.
 66. A method of detecting a chromosomal translocation ofa IB1 gene comprising the steps of: a) hybridizing a labeledpolynucleotide probe that specifically hybridizes with the IB1nucleotide sequence of SEQ ID NO:3 or its complement, to a chromosomespread from a cell sample to determine the pattern of hybridization andb) determining whether the pattern of hybridization differs from anormal pattern; whereby a difference in the pattern represents atranslocation.
 67. A method of detecting polymorphic forms ofRepro-EN-1.0 comprising the steps of: a) determining the identity of anucleotide or amino acid at a selected position within the sequence of atest Repro-EN-1.0 gene or polypeptide; b) determining the identity ofthe nucleotide or amino acid at the corresponding position of nativeRepro-EN-1.0 (SEQ ID NO:1 or 2) gene or polypeptide; and c) comparingthe identity from the test gene or polynucleotide with the identity ofthe native gene or polypeptide, whereby a difference in identityindicates that the test polynucleotide is a polymorphic form ofRepro-EN-1.0.
 68. A method of detecting polymorphic forms of IB1comprising the steps of: a) determining the identity of a nucleotide oramino acid at a selected position within the sequence of a test IB1 geneor polypeptide; b) determining the identity of the nucleotide or aminoacid at the corresponding position of native IB1 (SEQ ID NO:3 or 4) geneor polypeptide; and c) comparing the identity from the test gene orpolynucleotide with the identity of the native gene or polypeptide,whereby a difference in identity indicates that the test polynucleotideis a polymorphic form of IB1.